Diagnostics Subcommittee Report to the Tick-Borne Working Group

Co-Chairs: Monica E. Embers and Todd Myers

Information and opinions in this report do not necessarily reflect the opinions of the Working Group, the U.S. Department of Health and Human Services, or any other component of the Federal Government. Readers should not consider the report or any part of it to be guidance or instruction regarding the diagnosis, care, or treatment of tick-borne diseases or to supersede in any way existing guidance. All subcommittee members actively participated in the development of this report. Members voted to approve submission of the report to the Working Group and on the wording of each of the potential actions contained in the report. The vote to submit the report indicates general agreement with the content of the document, but it does not necessarily indicate complete agreement with each and every statement in the full report.

Background

Current Status of Diagnostic Testing for Tick-Borne Diseases in the United States 

Lyme disease and associated tick-borne infections currently constitute a public health crisis. In North America alone, the number of persons who are diagnosed with Lyme disease annually is estimated to be near half a million (Centers for Disease Control and Prevention, 2021a; Kugeler et al., 2021). Compounding the public health significance is the high rate of other tick-borne infections and co-infections, such as Babesia parasites, several species of Anaplasma, Ehrlichia, and Rickettsia bacteria, and several viruses. Given the limited sensitivity of the goldstandard diagnostic test for Lyme disease, the two-tier serologic test, new diagnostic tests are critically needed. The two-tier test’s lack of sensitivity in early infection is well established; therefore, the need for testing accuracy at all stages of disease is of the highest priority. Major challenges include the paucity of bacteria in the blood and other body fluids and variable antibody responses to infection, including possible seronegativity due to the capacity of Borrelia burgdorferi to suppress immune responses (Dattwyler et al., 1988; Tracy & Baumgarth, 2017).

Many new technologies have been applied to diagnostic testing for Lyme disease, and promising new assays are on the horizon. These include improvements to serologic tests, sensitive molecular detection, and “omics” approaches including metabolomics and immune profiles. Opportunities for personalized medicine include tick testing services, host genetic analyses, and prognostic indicators of disease or response to treatment. As early diagnosis of Lyme disease and other tick-borne diseases (TBDs) clearly results in improved outcomes, the investment in diagnostic testing should include increases in targeted Federal funding, improvements in the product development pipeline, and access to adequate patient samples for validation of novel diagnostic tests.

Critical to advancing diagnostic solutions for established and emerging TBDs is a better understanding of the diagnostic tools currently available and new technologies and approaches currently in development. Considering the current knowledge of risk factors for pathobiology of tick-borne pathogens, disease pathogenesis, approaches to establishing evidence of infection in patients, and the increased number of TBD cases in the United States, this subcommittee sought to assess the state of diagnostic science and commercial offerings, identify current and future strategies for increasing the sensitivity and specificity of detection, and examine barriers to the development and commercialization of diagnostic technologies and approaches. Importantly, the subcommittee focused on areas not fully addressed in previous Tick-Borne Disease Working Group subcommittees while building on the previous reports and recommendations. The new priorities identified are presented in detail in the Results and Potential Actions section of this report, which describes the priority issues and their importance, major challenges related to the priority issues, and potential actions to address these priorities.

Priority 1. Charge the Tick-Borne Disease Working Group to designate an Advisory Panel of experts in Lyme disease and tick-borne disease diagnostics to promote evaluation and development of current and promising new diagnostic approaches. The panel will serve to carry out the recommendations proposed in this report including Federal funding appropriations, scientific networking, and biorepository building.

Potential Action 1.1. The Tick-Borne Disease Working Group members will designate seven individuals to serve on the panel, under the Office of the Assistant Secretary for Health (OASH), HHS, comprised of stakeholders from the academic, clinical, industry, government, and advocacy sectors. This panel will convene from February 2023 through January 2025 with the goal of promoting the recommendations set forth in this report.

Potential Action 1.2. Fund the advancement of diagnostics for tick-borne disease through targeted requests for applications (RFAs), Small Business Innovation Research/Small Business Technology Transfer, Congressionally Directed Medical Research Programs, and other Federal funding mechanisms which are open to academic institutions and all industry entities. The funding opportunities should be inclusive of diagnostic tests intended to detect Lyme disease in all of its manifestations (early, disseminated, and chronic) and additional tick-transmitted infections.

Potential Action 1.3. Host an HHS-sponsored scientific conference to review promising proof-of-principle studies and applications of promising diagnostic technologies, including direct and indirect detection methods, as well as host immune biomarkers that may facilitate diagnosis. The conference should include researchers, government, investors, small businesses, large clinical labs, and diagnostics companies.

Potential Action 1.4. Mandate Federal funding for (1) the Centers for Disease Control and Prevention to build a national biorepository of human samples supported by a network of qualified labs and physician clinics and (2) fully fund the Center for Lyme Action’s proposed National Institutes of Health Centers of Excellence in Lyme and tick-borne disease to build a clinical research program involving a network of clinical academic centers with standardized case definitions and evaluation tools which will facilitate future research and development of diagnostic tests and outcome measures. Studies should cover prevention, diagnosis, and treatment interventions.

Priority 2. Educate policymakers, reviewers, researchers, and clinicians on the unique challenges of diagnostic test development and the innovation pipeline for tick-borne disease diagnostics.

Potential Action 2.1. Charge the National Academies of Sciences, Engineering, and Medicine (NASEM) to host webinars to educate researchers, doctors, and disease advocates on the latest science, working hypotheses, and future research needs.

Potential Action 2.2. Charge the Centers for Disease Control and Prevention to revise their web pages on tick-borne disease diagnostics in consultation with the Advisory Panel to reflect fully the advantages and limitations of currently recommended tests, and to provide more timely information about tests under development or not currently recommended.

Potential Action 2.3. Accelerate efforts by the Centers for Disease Control and Prevention to better engage the American public, clinicians, and relevant patient groups via an outreach program on the benefits of participating in clinical studies for tick-borne disease diagnosis. This is particularly important for patients to continue participating in longitudinal research to address the long-term effects of tick-borne disease and treatment efficacy.

Priority 3. Minimize roadblocks and streamline the process for getting new tick-borne disease diagnostics to market.

Potential Action 3.1. Expand the purview of the Biomedical Advanced Research and Development Authority (BARDA) to include vector-borne diseases and provide BARDA with funding to bring new tick-borne disease diagnostics to market. Build on existing initiatives to stimulate interest and funding for tick-borne disease diagnostic development and early commercialization, including multiphase funding for promising new diagnostic approaches.

Potential Action 3.2. Charge Federal entities (including the Centers for Medicare and Medicaid Services, U.S. Food and Drug Administration, Centers for Disease Control and Prevention, U.S. Department of Defense, and National Institutes of Health) to work with industry to streamline regulatory pathways and target solutions for getting new tick-borne disease diagnostic offerings to market. Federal entities should host healthcare summits focused on tick-borne disease including representatives from large commercial labs and diagnostics companies, regulatory agencies, insurers, medical researchers, providers, patients, and disease advocates on the innovation pipeline.

Potential Action 3.3. Charge Federal agencies to acknowledge that different testing methodologies may be required for different types and stages of Lyme disease, and other elusive tick-borne infections, similar to the model provided by tuberculosis guidelines. Focusing diagnostics solely on acute infection overlooks individuals affected by the full clinical spectrum of tick-borne disease.

Summary of State of the Science and Challenges to Diagnostic Advances

Diagnostic Testing for Multiple Tick-Borne Diseases and Potential for Serodiagnostic Test Improvement

Serologic assays (enzyme-linked immunosorbent assays [ELISAs] and western blots) that detect Borrelia-specific antibodies have become the standard tests used to diagnose Lyme disease. Following infection, antibodies develop usually within 28 days, but some may take as long as 6 weeks. Currently accepted for diagnosis of Lyme disease are (1) the two-tier test, consisting of an ELISA with confirmatory western blot, and (2) the modified two-tier test, consisting of a multi-antigen ELISA followed by a single antigen (C6) or other multi-antigen ELISA (Branda et al., 2011; Mead et al., 2019). Challenges to the reliability of these tests are the variable antibody responses among infected individuals, the unusual persistence of the IgM response, and the delay in detectable IgG levels, which reduces early Lyme disease sensitivity.

The blacklegged tick (Ixodes scapularis), also called the deer tick, is the most medically important arthropod in the United States. In addition to Lyme disease, at least four other serious illnesses are caused by infectious agents transmitted by this tick vector. These include babesiosis, anaplasmosis, B. miyamotoi infection, and Powassan virus infection, any of which may be severe or fatal in compromised hosts. Diagnostic testing is only available from a limited number of clinical laboratories, and methods are not standardized, partly owing to the absence of assays cleared by the U.S. Food and Drug Administration (FDA) for any of these infections. The strengths and limitations of available testing specific to each infection are presented in Table 1. Other medically important ticks include the American dog tick (Dermacentor variabilis), the lone star tick (Amblyomma americanum), the Gulf Coast tick (A. maculatum), and numerous others. Diagnosis of the myriad infections vectored by these ticks involves similar challenges as those enumerated in Table 1 for the blacklegged tick.

Opportunities include the use of serologic tests for diagnosis at later stages of disease, improving early test sensitivity with combined IgM/IgG testing, and the identification of antigen targets that induce the earliest antibody responses. Some evidence also indicates that broader antibody responses are also predictive of antibiotic treatment efficacy. New Luminex®-based assays utilizing borrelial peptides or whole proteins hold the promise of improving serodiagnostics beyond two-tier and modified two-tier testing (Blum et al., 2018; Radtke et al., 2021). Given the high prevalence of multiple pathogens in ticks, an opportunity exists not only in improving diagnostic tests for all TBDs, but also in testing ticks for specific pathogens post-exposure.

Vector Testing for Endemicity and Patient Exposure

An underutilized and often overlooked tool in the diagnostic toolbox is testing of the ticks themselves. Unlike mosquitoes, which bite quickly and then disperse, ticks may remain attached for up to 1 week to feed. This provides an opportunity to find and remove the tick before it has completed a blood meal. Through passive surveillance programs, experts can identify these medically relevant ticks and test them for pathogens to aid clinicians in coming to a clinical diagnosis should symptoms occur. Tick identification can be complicated, so a layperson or even a clinician can easily misidentify a tick and come to incorrect conclusions about which diseases may be associated with that tick bite (Kopsco et al., 2021). Direct molecular testing, such as polymerase chain reaction (PCR), can be used to detect viral and bacterial pathogens inside the tick. These pathogens can be abundant in the ticks themselves and are generally much easier to detect in the tick than in the host. Different pathogens are also transmitted from the tick to the host at different rates; engorgement status can be used to assess how long the tick has been attached and thus provide clues as to the likelihood of pathogen transmission.

Among others, local and state health departments, U.S. Department of Defense programs, and university-affiliated labs have set up successful tick testing services, which provide medically relevant information to tick-bite victims and their healthcare providers. These passive surveillance programs are also sources of a wealth of information regarding prevalence and distribution of ticks and tick-borne pathogens, especially as they change in response to shifting climate and urbanization. In combination with the published results of active surveillance, these datasets provide critical context to clinical decision makers so that emerging and endemic diseases are appropriately considered when making a diagnosis. The capacity to conduct these testing programs free of charge is largely limited by inconsistent funding (Mader et al., 2021). For maximum efficacy, these passive surveillance programs should be widespread, subsidized, and held to high-quality testing standards, such as confirming multiple genes for each pathogen or sequence-confirming results. Further, results must be confirmed quickly to aid in prompt diagnosis and should include information about the relative risk of infection attributed to that specific bite (Centers for Disease Control and Prevention, 2021b). Marrying conscientious tick testing programs with other diagnostic tools provides an opportunity to identify at-risk individuals before disease sets in and thus should be considered a useful component of personalized medicine for TBDs.

“Omics” Approaches

The application of “omics” approaches, which broadly fall under the headings of genomics, transcriptomics, proteomics, and metabolomics, have been promoted as potential approaches for the development of improved diagnostics for infectious disease, including Lyme disease. The power in these approaches comes from their ability to capture large quantities of molecular or biochemical data in an unbiased and untargeted manner. The primary concept behind the use of omics approaches is that unbiased omics data collection of specific molecular feature types (such as proteins, transcripts, or small molecules) can be exploited by machine learning and other computational approaches to define a minimal signature of host molecules that serves as a diagnostic biomarker of specific tick-borne infectious diseases. Further, the defined set of molecules that comprise the diagnostic biomarker can be measured using existing clinical laboratory technologies or emerging technologies that are tractable for clinical applications. In some cases, an omics technology or approach could possibly be utilized for direct detection of the infecting pathogen. Examples of this include metagenomic sequencing to detect pathogen genetic material or targeted/semi-targeted proteomics to detect pathogen-specific proteins or peptides. 

A wide variety of omics approaches have been investigated for use as potential platforms for tick-borne infectious disease diagnostics or to select panels of features that serve as diagnostic biomarkers. This includes transcriptomics (Bouquet et al., 2016; Petzke et al., 2020), multiple proteomics approaches (Douglas et al., 2011; Magni et al., 2015, 2020), metabolomics, and metagenomic sequencing (Kehoe et al., 2022; Pritt, 2021). Most of these studies have been directed at Lyme disease and most have served as proof of concept for their use. In at least one case, a specific proteomics approach is being applied as a CLIAbased diagnostic assay for Lyme disease (Magni et al., 2015).

Sensitive Molecular Testing for Direct Detection

An alternative to serology is testing aimed at direct detection of the pathogen. Direct detection tests have historically been performed using molecular assays such as PCR. These approaches have several advantages over serology: direct detection identifies an active infection, no lag period is necessary for the development of an antibody response, and multiplex assays have the capacity to test for more than one agent. The downside of molecular testing is that this approach has not been useful for the diagnosis of Lyme disease, primarily because of transient and limited quantity of bacteria in blood.

Within the past decade, the advent and widespread implementation of next-generation sequencing (NGS) has provided a unique opportunity to overcome the previous limitations of molecular testing. Unbiased NGS detection tests facilitate simultaneous detection of all agents in a clinical sample while employment of agent-specific oligonucleotide probes for enrichment of desired nucleic acids vastly improves assay sensitivity and provides a detection capability far superior to PCR (Briese et al., 2015; Jain et al., 2021). In addition, technological advancements have resulted in a decrease in costs, labor, and length of time required for NGS data generation and analyses (Gu et al., 2021). The development of portable sequencers has created the potential to establish NGS as a field-deployable frontline platform, and perhaps even as point-of-care testing in the future (Smith et al., 2020).

The development and recent application of droplet digital PCR (ddPCR) also offers strong potential for overcoming the limitations and low sensitivity of PCR testing, especially in combination with sample enrichment methods (King et al., 2017; Maggi et al., 2020, 2021; Wilson et al., 2015). While ddPCR equipment and reagent costs are currently quite high, the costs will come down over time as clinical applications increase, regulatory standards are established, and insurance reimbursements are determined. Recent advances in ddPCR technology, such as the Bio-Rad QX ONE ddPCR System, could provide additional affordability in the form of multiplex assays in which multiple infections could be confirmed in a single patient sample (Maggi et al., 2021).

Opportunities for Personalized Medicine

Personalized medicine can be provided in many forms. TBDs, with their variance in response to infection and disease presentation between individuals, are an opportune example of how personalized medicine can be utilized. These opportunities may include prognostic indicators of disease resolution and antibiotic efficacy, determination of genetic predisposition for specific manifestations of disease, and immune correlates of acute versus chronic disease.

One example of how genomics can be used as a support tool for clinicians comes from work with autism spectrum disorders. By the analyses of single nucleotide polymorphisms (SNPs), deletions, or mutations in genes, information can be gleaned from the affected metabolic, developmental, or immunologic pathways (Way et al., 2021). The affected genes can then be modulated with natural or synthetic therapeutics to provide balance in the pathways leading to disease or dysfunction. While much is yet to be learned of how genomics can inform treatment for Lyme disease and other TBDs, the association has been made between variation (a specific combination of SNPs) in the ABCB1 transporter gene and the development of post-treatment Lyme disease (PTLD) (Lyon & Seung, 2019). With respect to immune function, SNPs of toll-like receptors and interleukin-6 promoter have also been linked to persistent symptomatology and disease (Hein et al., 2019; Strle et al., 2012).

Another example of research to enable personalized medicine includes the identification of biomarkers for disease states. In one study, chemokines and cytokines in the serum of patients were quantified in a multiplex platform, demonstrating differences that correlated with disease states. Specifically, elevation in the chemokine CCL19 after treatment of early Lyme disease has been associated with the later development of PTLD (Aucott et al 2016; Soloski et al., 2014).

Current Availability of Patient Samples for Testing and Validation

Well-characterized human biologic samples are vital for conducting research. This is particularly true for developing and validating diagnostic tests for TBDs. When using samples in their assays, test developers need to understand what types of patient samples were collected, including the following information: signs and symptoms of disease and other relevant health history; what biologic materials were collected; when these materials were collected in the course of disease; how samples were processed and stored; and how samples where characterized, including any PCR, serology, or other laboratory testing that was performed.

In addition to benefiting diagnostic test developers and researchers, well-characterized sample repositories also benefit medical providers, patients, and the greater public at risk for Lyme disease (Molins et al., 2014). Currently, three established Lyme disease repositories exist, including the Centers for Disease Control and Prevention (CDC) Lyme Serum Repository (Molins et al., 2014), the Lyme Disease Biobank (Horn et al., 2020), and the Study of Lyme disease Immunology and Clinical Events (SLICE) at the Johns Hopkins Lyme Disease Research Center (Rebman et al., 2015). Additionally, some investigators have their own sample collections. Each biorepository has distinct inclusion and exclusion criteria and sample characterization criteria. While well-characterized Lyme disease patient samples are available, more are needed because the samples in these repositories will need to be replenished as they are depleted by investigators working on Lyme disease. For other tick-borne infections, very limited well-characterized samples are available, particularly for those that are rare. 

Challenges to Novel Test Commercialization

Commercialization of diagnostics through the current regulatory review (CLIA for lab-developed tests and FDA for medical devices; Testing.com, 2021a, 2021b) and test adoption is a complicated process, fraught with obstacles. It involves many steps, including clinical research, publication in medical guidelines, physician education, and enabling insurance coverage. Moreover, the process is prohibitively expensive with considerable economic disincentives for investors (Faruki & Lai-Goldman, 2010). Time to sustainable market adoption can take decades, and cost ranges from $31 million to $94 million per test application (Kirsch, 2019) with no guarantee of a return on investment.

Importantly, we are already facing a shortage of testing innovations in the United States, and calls for stricter regulation, such as the Verifying Accurate, Leading-edge IVCT Development (VALID) Act, could further interfere with access to new and better testing (Shirts, 2020). Doctors are very concerned about regulatory agencies interfering with the practice of medicine. At this time, the FDA approval pathway is especially problematic for new and rare diseases, such as many TBDs for which the market size may not justify the investment, and for diseases for which test designs must remain customizable or flexible.

Prior Tick-Borne Disease Working Group reports called specifically for development of FDA-approved tests, but not all tests are suitable for scale as a medical device (in vitro diagnostics [IVD] or test kit) or testing equipment. Historically, the bulk of clinical testing in the United States belongs to the category of lab-developed tests (Pew, 2021). FDA will sometimes review and certify lab-developed tests, but this is not a common occurrence and can significantly increase the costs of commercialization. The lab-developed test pathway allows the development and commercialization of laboratory testing as a service in which doctors interpret the results. However, this regulatory path to market comes with its own problems in terms of variable levels of published scientific evidence for new test methods, the need for clinician and patient guidance to support proper adoption and clinical use, and the lack of transparency around certain labs offering new TBD test options.

Regulatory uncertainty, in addition to the political controversy surrounding TBD testing, creates huge disincentives for investors, physicians, patients, insurance companies, and, frankly, researchers and diagnostic companies to advance TBD testing. The unfortunate result is that the most promising diagnostic advances become stuck in the innovation pipeline, failing to move out of the research lab and into clinical practice or stalling at an early stage of clinical proof as a lab-developed test. Navigating such a complex and contentious marketplace is difficult for healthcare consumers and providers, and commercializing new diagnostic technologies in compliance with best practices in diagnostic innovation is onerous for medical labs. These challenges are further complicated by crude efforts to provide consumers with guidance on TBD test recommendations, for example, by lumping all novel diagnostic approaches, regardless of published evidence, in false opposition to a historically recommended standard of diagnostic care (Centers for Disease Control and Prevention, 2018).

Indeed, commercializing new TBD diagnostic technologies under a broadly focused direct-to-consumer business model (U.S. Food & Drug Administration, 2019) or with splashy marketing and broad claims of FDA-reviewed transformative innovation (for example, the Theranos case) (Rutschman, 2021) is much easier than commercializing new clinical tests as lab-developed tests available with a doctor’s order. The research and development and early commercialization teams that are publishing clinical evidence of validation and utility and working steadily on clinical evidence, medical education, and insurance coverage need governmental support to ensure that patients and their doctors have access to the best TBD diagnostic tools available.

This report, developed by the Diagnostics Subcommittee of the Tick-Borne Disease Working Group, outlines priority issues and potential actions aimed at tackling the many diagnostic challenges associated with TBDs. The subcommittee aims to generate opportunities that will pave the way for innovative solutions, assist clinicians in the treatment of their patients, and translate to significantly improved outcomes for individuals affected by TBDs.

Methods

Characteristics of the Subcommittee

At the Tick-Borne Disease Working Group Public Meeting on August 26, 2021, Monica Embers and Todd Myers volunteered to serve as co-chairs for the Diagnostics Subcommittee. They then formed a subcommittee of two Federal and eight public representatives, offering the perspectives and expertise of multiple stakeholder groups (see Table 2: Members of the Diagnostics Subcommittee).

Subcommittee Meetings

The subcommittee held 16 2-hour virtual meetings (see Table 3: Overview of Diagnostics Subcommittee Meetings). During this time, members:

• Considered relevant literature and public comments
• Reviewed current Federal activities
• Identified gaps in knowledge and areas where Federal funding is needed
• Heard from 22 expert speakers (see Table 4: Presenters to the Diagnostics Subcommittee)
• Generated their report to the Tick-Borne Disease Working Group

Public Comment and Inventory

Public comments were submitted between November 2020 and September 2021 to the Tick-Borne Disease Working Group mailbox for review. The Public Comment Subcommittee reviewed these emails and provided relevant comments to the Diagnostics Subcommittee. The subcommittee co-chairs reviewed the comments and ensured the public’s concerns were represented in the priorities and potential actions. Specific topics identified by members of the public include the following:  

• Formation of a New Hampshire Lyme Disease Study Commission devoted to current diagnostics assessment, TBD incidence, and physician and public education
• Emphasis on the significant individual (and national) economic impact of unreliable diagnostic testing
• Request for patient-focused, actionable recommendations with accountability
• The importance of advancing understanding of how Lyme disease causes chronic illness
• The need for National Institutes of Health (NIH) research (including patient and provider input) on diagnosis and treatment of late Lyme disease and persistent illness with a focus on reducing treatment failures for both late and early Lyme disease
• Call for improved testing, awareness campaigns, practitioner and public health education, and data that reflect the community of Lyme disease patients
• The challenge of surveillance data that are incongruous with findings from independent patient studies (for example, those of Lymedisease.org) and the notion that TBDs are no longer limited to certain regions of the country

Subcommittee Report Development

During the Public Meeting on August 26, 2021, the Tick-Borne Disease Working Group identified the following topics to be considered by the Diagnostics Subcommittee:

• All TBDs
• New test development and validation
• Gaps and limitations in current testing
• Promising techniques on the horizon, including real-time tests
• Approval and standardization processes
• Deeper dive into the roadblocks for better testing
• Testing for surveillance

The subcommittee identified expert speakers to present information on those topics. As the meetings progressed, members identified priority themes upon which to focus their report. Within each theme, they identified concrete potential actions the Federal government can undertake to advance key diagnostic issues. Each member then volunteered to write sections of the report based on their expertise.

Brief for the Working Group

At Public Meeting 20 on February 28 and March 1, 2022, the subcommittee co-chairs presented the final report to the Tick-Borne Disease Working Group using a PowerPoint template provided to them by the leadership and support team. The presentation was finalized after subcommittee members were given the opportunity to provide feedback via email.

Table 4: Presenters to the Diagnostics Subcommittee

Meeting No.	Presenter(s)	Topics Addressed
2	Liz Horn, PhD, MBI, Lyme Disease Biobank	Lyme Disease Biobank
3	Monica Embers, PhD, Division of Immunology, Tulane National Primate Research Center	Lyme Disease Diagnostics
3	John A. Branda, MD, Massachusetts General Hospital, Harvard Medical School	Update on Serologic Testing for Selected Tick-Borne Diseases
3	Robyn M. Nadolny, PhD, Molecular Biology Section, Laboratory Sciences Directorate, U.S. Army Public Health Center	Tick Testing: A Tool for Tick-Borne Disease Surveillance and Diagnostics
4	Timothy Haystead, PhD, Duke University School of Medicine	Live Imaging Diagnostics and Molecularly Targeted Therapies for Borrelia
4	John T. Belisle, PhD, Department of Microbiology, Immunology, and Pathology, Colorado State University	Lyme Disease Diagnostics: Metabolomics
5	Rafal Tokarz, PhD, Center for Infection and Immunity, Columbia University	Enhanced Detection of Tick-Borne Agents Using Capture Sequence
5	Amanda Elam, PhD, Galaxy Diagnostics	Commercialization Pathway (Introduction)
6	Sudeb Dalai, MD, PhD, Adaptive Biotechnologies; Infectious Diseases, Stanford University School of Medicine	Immunosequencing of the T-cell Receptor Repertoire Reveals Signatures for Diagnosis and Characterization of Early Lyme Disease
7	John Aucott, MD, Johns Hopkins University School of Medicine	Diagnosis of Acute Lyme Disease
7	Laimonas Kelbauskas, PhD, and Neal Woodbury, PhD, Arizona State University	Using Antibody Profiling and Machine Learning to Discover Novel Biomarkers for Lyme Disease
8	Brandon Jutras, PhD, Fralin Life Sciences Institute, Virginia Tech	Using the Unusual Peptidoglycan Cell Wall of Borrelia burgdorferi to Diagnose Lyme Disease
8	Ricardo Maggi, PhD, Intracellular Pathogens Research Laboratory, North Carolina State University	Bartonella-Borrelia-Babesia Droplet Digital PCR Assay (BBB-ddPCR)
9	Lance A. Liotta, MD, PhD and Alessandra Luchini, PhD, George Mason University	Nanotechnology-Enabled Mass Spectrometry Reveals Tick-Borne Pathogen-Specific Urinary Peptides
9	Amanda Elam, PhD, Galaxy Diagnostics	Advancing Diagnostics for Tick-Borne Diseases: Demystifying the Commercialization Pipeline
10	Melanie K. B. Wills, PhD, G. Magnotta Lyme Disease Research Lab, University of Guelph	Targeting Lyme Disease: The Quest for a Diagnostic Bull’s-Eye
10	Jacob Lemieux, MD, DPhil, Massachusetts General Hospital, Harvard Medical School, The Broad Institute	Sequence-Based Diagnosis of Tick-Borne Pathogens
11	Mary M. Petzke, PhD, New York Medical College	Gene Expression Biomarkers for the Diagnosis of Lyme Disease
11	Charles Chiu, MD, PhD, University of California, San Francisco	Overcoming Diagnostic Challenges in COVID-19 and Lyme Disease
13	Hari Krishnamurthy, PhD, Vibrant Sciences; Daniel A. Green, MD, and Alex J. Rai, PhD, Columbia University	Innovative Approaches to Tickborne Disease Diagnostics
13	Sharon Hausman-Cohen, MD, IntellxxDNA, Resilient Health	Genomics as a Clinical Decision Support Tool
14	Christopher Houchens, PhD, Biomedical Advanced Research and Development Authority (BARDA)	How BARDA Supports the Accelerated Research, Development, and Delivery of Medical Countermeasures Against Public Health Threats (pre-recorded)

Table 5: Votes Taken by the Diagnostics Subcommittee

Meeting Number	Motion	Result Result In Favor	Result Opposed	Result Abstained	Result Absent	Minority Response
By email	Approve Background	8	0	0	2	None
By email	Approve Priority 1	9	0	0	1	None
16 and by email	Approve Potential Action 1.1	8	0	0	2	None
16 and by email	Approve Potential Action 1.2	8	0	0	2	None
16 and by email	Approve Potential Action 1.3	8	0	0	2	None
16 and by email	Approve Potential Action 1.4	8	0	0	2	None
16 and by email	Approve Priority 2	9	0	0	1	None
16 and by email	Approve Potential Action 2.1	8	0	0	2	None
16 and by email	Approve Potential Action 2.2	8	0	0	2	None
16 and by email	Approve Potential Action 2.3	8	0	0	2	None
16 and by email	Approve Priority 3	9	0	0	1	None
16 and by email	Approve Potential Action 3.1	8	0	0	2	None
16 and by email	Approve Potential Action 3.2	8	0	0	2	None
16 and by email	Approve Potential Action 3.3	6	2	0	2	None
By email	Approve full report, including additional copyediting	8	0	0	2	None

Results and Findings

For consideration by the Tick-Borne Disease Working Group, the Diagnostics Subcommittee has identified three major priorities and 10 potential actions to achieve them.

Priority 1. Charge the Tick-Borne Disease Working Group to designate an Advisory Panel of experts in Lyme disease and tick-borne disease diagnostics to promote evaluation and development of current and promising new diagnostic approaches. The panel will serve to carry out the recommendations proposed in this report including Federal funding appropriations, scientific networking, and biorepository-building.

Background

Two prior subcommittees have prioritized the development of promising new diagnostic tests for TBDs. During the period between the first subcommittee report (2018) and this one, many promising new tests have been developed and are now at various points in the commercialization pipeline. As our meeting discussions indicated, there is a disconnect between product development, testing and validation, and commercial development. As such, we propose to ensure follow-through of recommendations by prioritizing the formation of an Advisory Panel to facilitate and aid in enacting the priorities and potential actions put forth in this 2022 report.

Summary of Evidence and Findings

This subcommittee heard from 22 experts whose research has focused on improving the diagnosis of Lyme disease and other TBDs. A multitude of different technologies were highlighted, each of which had benefits over the current standard for testing. These novel strategies merit the opportunity to continue evaluation, validation, and, if warranted, commercialization. In other words, this subcommittee did not find that there was an absence of new technologies or interest, but rather that the investment in the future of these tests is a major gap hindering the transition of novel research findings into improved patient outcomes.

Challenges

Notable challenges include the relative scarcity of well-characterized patient samples to be used for test evaluation and validation, the lack of standardization of diagnostic tests (evaluation of different tests in different labs), and appropriate comparators for tests utilizing different platforms. Test performance is most frequently assessed using acute phase patient samples, leaving limited diagnostic utility for different stages of infection and different disease presentations.

Opportunities

Many of the new testing modalities proposed for Lyme disease and other TBDs have precedence with use in other infectious diseases. Developers have acquired samples from the Lyme Disease Biobank, among other sources. The tests should be vetted with a large variety of patient samples from those with early acute infection to those who have chronic symptoms. The opportunity to acquire small business grants should be utilized and this program can be expanded. Finally, bringing together stakeholders would offer the benefit of sharing successes and failures, which may accelerate development. The funding and creation of Centers of Excellence focused on TBD patient outcomes and clinical research could be a critical component of overcoming hurdles for improved diagnostics.

Priority 1 Potential Actions

Potential Action 1.1. The Tick-Borne Disease Working Group members will designate seven individuals to serve on the panel, under the Office of the Assistant Secretary for Health (OASH), HHS, comprised of stakeholders from the academic, clinical, industry, government, and advocacy sectors. This panel will convene from February 2023 through January 2025 with the goal of promoting the recommendations set forth in this report.

Table 6: Vote on Potential Action 1.1

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 1.2. Fund the advancement of diagnostics for tick-borne disease through targeted requests for applications (RFAs), Small Business Innovation Research/Small Business Technology Transfer, Congressionally Directed Medical Research Programs, and other Federal funding mechanisms which are open to academic institutions and all industry entities. The funding opportunities should be inclusive of diagnostic tests intended to detect Lyme disease in all of its manifestations (early, disseminated, and chronic) and additional tick-transmitted infections.

Table 7: Vote on Potential Action 1.2

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 1.3. Host an HHS-sponsored scientific conference to review promising proof-of-principle studies and applications of promising diagnostic technologies, including direct and indirect detection methods, as well as host immune biomarkers that may facilitate diagnosis. The conference should include researchers, government, investors, small businesses, large clinical labs, and diagnostics companies.

Table 8: Vote on Potential Action 1.3

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 1.4:  Mandate Federal funding for (1) the Centers for Disease Control and Prevention to build a national biorepository of human samples supported by a network of qualified labs and physician clinics and (2) fully fund the Center for Lyme Action’s proposed National Institutes of Health Centers of Excellence in Lyme and tick-borne disease to build a clinical research program involving a network of clinical academic centers with standardized case definitions and evaluation tools which will facilitate future research and development of diagnostic tests and outcome measures. Studies should cover prevention, diagnosis, and treatment interventions.

Table 9: Vote on Potential Action 1.4

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Minority Responses

There were no minority responses.

Priority 2. Educate policymakers, reviewers, researchers, and clinicians on the unique challenges of diagnostic test development and the innovation pipeline for tick-borne disease diagnostics.
Background

In the two previous Diagnostics Subcommittee reports (2018 and 2020), the need for better testing for TBDs was clearly stated, especially direct testing and FDA-approved testing. While testing advances have occurred, few have reached the point of commercialization, most are indirect methods that do not represent any improvement in sensitivity, and few involve direct detection. An improved understanding is needed of the challenges of advancing diagnostics for TBDs, from the technical challenges presented by the pathobiology of each pathogen to those inherent in the development and commercialization of any new diagnostic approach for new and rare diseases.

Summary of Evidence and Findings

Several peer-reviewed publications describe recent advances in both serologic and direct detection methods (Arumugam et al., 2019; Eshoo et al., 2012; Jain et al., 2021; Jayaraman et al., 2020; Joung et al., 2020). However, other diagnostic solutions have been commercialized that do not have any published data to support clinical performance or demonstrated utility for acute or later stage infections (Branda et al., 2018; Schutzer et al., 2019).

Challenges

Lack of funding has made it very difficult for scientists to focus time and energy on the development of advanced techniques. Few individuals who serve as reviewers have much knowledge of the unique pathobiology of Borrelia and other tick-borne pathogens, and the funding pools tend to be small and highly competitive for both Federal and foundation-based grant sources. Of the few technologies that have reached the prototype stage, only a few have made it to the first stage of commercialization as a lab-developed assay and only one has made it to the stage of FDA-approved IVD kit (AACC, 2019). Importantly, few incentives for investors to support these testing advances and even less understanding of the unique challenges presented by TBD infections exist. The controversy over new potential diagnostic solutions further damages the credibility of potential solutions and the scientists who have pursued innovation in this highly contested clinical marketplace. Some commercial labs have chosen to sidestep the rigorous pathway to establish clinically proven diagnostic methods through direct-to-consumer testing. The result is that clinically proven approaches are not only hobbled in the development process, but also may be discredited by medical experts along with assays lacking a clear evidence base.

Opportunities

Educating medical experts, disease advocates, and consumers on the development and commercialization pathway could help clarify which technologies and diagnostic solutions hold the most potential for supporting a more accurate diagnosis of disease. For example, CDC could replace its current “Lab Tests Not Recommended” web page with a page that provides guidance to clinicians and consumers on the types of technologies that are in the innovation pipeline with different levels of evidence.

The controversy resulting from the aptly named “Lyme Wars” complicates both the development and commercialization of new diagnostic approaches by discouraging investors from investing, clinicians from ordering, and patients from participating in clinical studies. Educating the American public on the importance of both clinical evidence and the benefits as well as the risks of participating in clinical studies would help establish biobanks for research and clinical studies to clarify clinical utility of different diagnostic approaches, which will speed our way toward new and actionable clinical understandings of these important emerging diseases.

Priority 2 Potential Actions

Potential Action 2.1. Charge the National Academies of Sciences, Engineering, and Medicine (NASEM) to host webinars to educate researchers, doctors, and disease advocates on the latest science, working hypotheses, and future research needs.

Table 10: Vote on Potential Action 2.1

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 2.2. Charge the Centers for Disease Control to revise their web pages on tick-borne disease diagnostics in consultation with the Advisory Panel to reflect fully the advantages and limitations of currently recommended tests, and to provide more timely information about tests under development or not currently recommended.

Table 11: Vote on Potential Action 2.2

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 2.3. Accelerate efforts by the Centers for Disease Control and Prevention to better engage the American public, clinicians, and relevant patient groups via an outreach program on the benefits of participating in clinical studies for tick-borne disease diagnosis. This is particularly important for patients to continue participating in longitudinal research to address the long-term effects of tick-borne disease and treatment efficacy.

Table 12: Vote on Potential Action 2.3

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Minority Responses

There were no minority responses.

Priority 3. Minimize roadblocks and streamline the process for getting new tick-borne disease diagnostics to market.

Background

Despite recent progress in the development of TBD diagnostics, few innovations are advancing beyond the research lab into commercial laboratories. Those advances that are currently offered for clinical use too often remain stalled at the first stage of commercialization as lab-developed tests while stakeholders invest against all odds in clinical research, peer-review publication, medical education, and insurance reimbursement review process. Few diagnostic solutions progress beyond lab services to FDA review and approval as IVD kits because of the lack of understanding of the true prevalence and disease burden of tick-associated diseases, which results in poor funding for development and commercialization.

Summary of Evidence and Findings

Recent analyses of funding for Lyme disease suggests that the majority of Federal funding has gone to basic (versus translational) research at a small number of institutions. Funding allocations can lag behind current disease burden by as many as 10 years (Ballreich et al., 2021), and the financial burden of Lyme disease in both the United States and Europe is staggering (Adrion et al., 2015; Mac et al., 2019). Importantly, as is the case for several other diseases, Federal funding does not correlate with disease burden; disproportionate allocations of NIH funding have gone to HIV, malaria, Ebola, Zika, and, most recently, COVID-19.

Challenges

TBDs compete directly with other infectious diseases, many of which currently have much lower or no incidence in the United States. The global SARS-CoV-2 pandemic has unfortunately, but necessarily, detracted funding for endemic or chronic infectious diseases and infections with high morbidity but lower mortality.

Opportunities

Creating a national initiative to stimulate interest and funding for TBD diagnostic test development is imperative to ensure that these diseases are addressed independently from infections that kill quickly, such as hospital-acquired infections, or those that affect vast populations, such as influenza and novel coronaviruses. Several initiatives designed to draw interest and investment in advancing diagnostic solutions for TBD are underway, such as the LymeX Innovation Accelerator public-private partnership and the CDC-led national framework for TBD. Funds could be allocated to build on the foundation of these programs to more rapidly expand awareness and investments in advancing diagnostics to support both research and clinical use.

Priority 3 Potential Actions

Potential Action 3.1. Expand the purview of the Biomedical Advanced Research and Development Authority (BARDA) to include vector-borne diseases and provide BARDA with funding to bring new tick-borne disease diagnostics to market. Build on existing initiatives to stimulate interest and funding for tick-borne disease diagnostic development and early commercialization, including multiphase funding for promising new diagnostic approaches. 

Table 13: Vote on Potential Action 3.1

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 3.2. Charge Federal entities (including the Centers for Medicare and Medicaid Services, U.S. Food and Drug Administration, Centers for Disease Control and Prevention, U.S. Department of Defense, and National Institutes of Health) to work with industry to streamline regulatory pathways and target solutions for getting new tick-borne disease diagnostic offerings to market. Federal entities should host healthcare summits focused on tick-borne disease including representatives from large commercial labs and diagnostics companies, regulatory agencies, insurers, medical researchers, providers, patients, and disease advocates on the innovation pipeline.

Table 14: Vote on Potential Action 3.2

Number in Favor	Number Opposed	Number Abstained	Number Absent
8	0	0	2

Potential Action 3.3. Charge federal agencies to acknowledge that different testing methodologies may be required for different types and stages of Lyme disease, and other elusive tick-borne infections, similar to the model provided by tuberculosis guidelines. Focusing diagnostics solely on acute infection overlooks individuals affected by the full clinical spectrum of tick-borne disease.

Table 15: Vote on Potential Action 3.3

Number in Favor	Number Opposed	Number Abstained	Number Absent
6	2	0	2

Minority Responses

There were no minority responses.

Discussion and Big Picture Summary

The Diagnostics Subcommittee findings indicate that there does not appear to be a paucity of novel ideas or technologies that are intended to improve diagnostic testing for Lyme disease and other TBDs. Rather, the path to product development and commercialization is stifled by a lack of funding and support. Using the analogy of NIH small business grant funding mechanisms, many of these candidate diagnostic tests are stuck in the Phase I (optimization, development, and testing) stage. Thus, more investment needs to be made in the Phase II (path to commercialization) stage. Widespread agreement exists that the currently approved gold standard test (two-tier serology) is not good enough. Many advanced diagnostic testing platforms are already showing improvement over two-tier testing. While there may never be a single one-size-fits-all test developed to diagnose Lyme disease and associated TBDs, we can certainly do better than what is currently approved, and that may require us to consider multiple tiers if necessary.

Appendix

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