Background Issues and Priorities Methods Results Discussion References Appendicies 1-10 Disclaimer: 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; and they are not intended to be medical advice or to serve as a medical resource for patient care or drug use or drug dosing. Background The work of our committee is focused on the transmission, pathogenesis, and treatment of Lyme disease. The CDC estimates that more than 300,000 new cases of Lyme disease occur annually in the United States, each requiring safe and effective antibiotic treatment (Hinckley et al., 2014). The fundamental goal of antibiotic treatment for Lyme disease is to restore health by eliminating the disease-causing pathogens. Yet many patients have ongoing symptoms or signs of the illness despite prior antibiotic therapy (Aucott, Rebman, Crowder, & Kortte, 2013; Fallon et al., 2008; Klempner et al., 2001; Krupp et al., 2003; Shadick et al., 1994). The exact percentage of these patients is unknown and the risk for antibiotic treatment failure appears to correlate with the duration of the infection such that patients treated earlier in the illness fare better than those whose treatment was delayed (Aucott, Crowder, & Kortte, 2013; Logigian, Kaplan, & Steere, 1999; Maloney, 2016). Patients with longstanding untreated disease and those with ongoing manifestations may experience short- and long-term disabilities, and the attendant financial and societal burden can be significant (Adrion, Aucott, Lemke, & Weiner, 2015; Johnson, Aylward, & Stricker, 2011). Thus, it is crucial to identify highly effective therapies to shorten the duration of the illness and to minimize the number of people who remain ill following treatment. Identifying highly effective treatments requires a foundational understanding of B. burgdorferi, the bacterial agent of Lyme disease, including the processes that 1) facilitate its transmission to humans, 2) permit the establishment of infection in the immediate post-bite period, and 3) maintain the infection in animal species and humans. It also requires an understanding of the pathophysiology underlying the various symptoms and signs of infection and the identification of clinical markers that portend a higher risk of treatment failure, as this could lead to novel therapeutic approaches for such individuals. The etiology of persistent symptoms and signs of illness after infection with B. burgdorferi are unknown and may be multi-factorial. Identifying the underlying pathophysiology of illness and developing markers distinguishing persistent disease mechanisms could lead to the establishment of appropriately directed therapies. Clinical advances in this area could reduce patient suffering, restore health, and limit the financial and societal burdens associated with ongoing disease. Major challenges/issues The challenges in our areas of concern are many. The understanding of the pathogen and the basic elements that contribute to disease are incomplete. The interaction between B. burgdorferi and different components of the immune system have not been fully investigated, leaving many gaps in our understanding of pathogen transmission and maintenance in mammals. It is necessary here to explain our usage of the term “persistence,” as the definition is foundational to the materials we compile herein. Multiple studies, in vitro, in animal models, and in humans, have shown that B. burgdorferi can survive long term in the presence of a mammalian immune response (Armstrong, Barthold, Persing, & Beck, 1992; Barthold, 1993; Barthold, Persing, Armstrong, & Peeples, 1991; Baum, Hue, & Barbour, 2012; Elsner, Hastey, Olsen, & Baumgarth, 2015; Embers et al., 2012; Schaible, Kramer, Justus, Museteanu, & Simon, 1989; Steere, 2001; Zhang, Mathiesen, et al., 1997; Zhong et al., 1997). The mechanisms for persistence are likely closely linked to the ability of B. burgdorferi to evade host immunity, as shown in numerous studies, conducted mostly in mice. These studies have shown that B. burgdorferi employs various immune evasion mechanisms, including the up- and down-regulation or molecular changes to various surface expressed outer surface proteins (Coutte, Botkin, Gao, & Norris, 2009; Norris, 2006), as well as mechanisms to reduce the functionality of the innate immune response (Miller, Ma, Crandall, Wang, & Weis, 2008), including the inhibition of complement activation (Garcia, Zhi, Wager, Hook, & Skare, 2016; Kraiczy, 2016a), as well as the adaptive immune response (Elsner et al., 2015; Fikrig et al., 1996; Hastey, Elsner, Barthold, & Baumgarth, 2012; Tracy & Baumgarth, 2017). Thus, existing data indicate that B. burgdorferi survives in otherwise immunocompetent hosts by reducing the effectiveness of the immune response. In addition, numerous studies have shown the ability of B. burgdorferi to survive antibiotic therapy in vitro, in animals, and in humans ADDIN EN.CITE.DATA (Aucott, Crowder, et al., 2013; Aucott, Rebman, et al., 2013; Fallon et al., 2008; Feng, Auwaerter, & Zhang, 2015; Hinckley et al., 2014; Hodzic, Feng, Holden, Freet, & Barthold, 2008; Klempner et al., 2001; Krupp et al., 2003; Logigian et al., 1999; Nocton et al., 1994; Oksi, Marjamaki, Nikoskelainen, & Viljanen, 1999; Preac-Mursic et al., 1989; Shadick et al., 1994; Sharma, Brown, Matluck, Hu, & Lewis, 2015; Straubinger, 2000; Strle et al., 1993). It is therefore likely that multiple mechanisms contribute to the bacteria’s ability to survive both the mammalian immune response and antibiotic therapy. Thus, we will use the broad definition for “persistence” to describe the survival in both antibiotic-naïve and antibiotic-treated mammals, as the mechanisms causing persistence in these cases may in fact overlap. The spectrum of disease manifestations in both antibiotic-naïve and antibiotic-treated patients is quite broad and the correlation between the pathophysiology and individual disease manifestations requires additional study. While several mechanisms have been proposed, detailed assessments of each have yet to be completed. There have been few human clinical treatment trials with B. burgdorferi-infected patients, and those conducted have enrolled small numbers of patients. This lack of high-quality human trial data hampers our ability to identify optimal treatment strategies for the different stages and manifestations of the infection in patients. In addition, the small enrollment numbers of human trials have precluded our ability to identify and differentiate among subgroups of patients, for example, those who are treatment responders and those who are not. The absence of reliable and easily obtained biomarkers of the infection complicate trial design, as it can currently not be established whether subjects are actively infected with B. burgdorferi at enrollment. Similarly, absent a reliable test for B. burgdorferi eradication, it is difficult to determine treatment efficacy and therefore difficult to develop optimal treatment approaches. In cases where symptoms and signs of disease continue following treatment, it is difficult to know if they represent persistent infection or other potential mechanisms of disease, which also lack biomarkers. The inability to identify and then eliminate specific after-treatment disease mechanisms limits the prospects for developing and effectively delivering mechanism-directed therapies. This report discusses key issues in pathogenesis, transmission, and treatment of Lyme disease. We review the evidence regarding mechanisms of the pathogen B. burgdorferi that allow it to establish and cause persistent infection in mammalian hosts. We discuss the pathogenesis of persistent disease symptoms in antibiotic-naïve and antibiotic-treated patients and previously conducted clinical trials for various clinical presentations. Furthermore, we also identify gaps in our understanding that may impact patient care, including the lack of biomarkers to detect infection and bacterial eradication; limited research on the effects of delayed diagnosis, immune dysfunction, persistent infection, coinfections, and neural dysregulation on ongoing disease symptoms. Finally, we discuss the need for a variety of clinical research tools that can measure patient-centered outcomes and the role of the patient in medical decision-making. Issues and Priorities Our subcommittee covers pathogenesis, treatment, and transmission of tick-borne diseases. Like other subcommittees, we will cover broad swaths of contents that touch on many areas important to all stakeholders – patients, physicians, researchers, and government bodies alike. Our subcommittee includes members from these different groups, which has made our discussions robust because of the different perspectives provided. Early on in our discussions, the group decided to focus on B. burgdorferi in the first report to the Working Group. There is a basic understanding that other infections transmitted by ticks may play a major role in the difficulty of diagnosing and treating patients who present to doctors with tick-borne infection, either alone or in combination with B. burgdorferi. However, the group felt that the short time frame did not allow for discussion on other tick borne-diseases under the pathogenesis, treatment, and transmission umbrella. During our first breakout call, group brainstorming brought out several key themes of interest. First, the concept of persistence of B. burgdorferi emerged as the commonly mentioned important topic for study. Notably the definition of persistence can be different in different circumstances, and we will explain our definition in the Background section. The need for study of pathophysiology of and treatment for both antibiotic-naïve and antibiotic-treated patients was also discussed. Biomarkers to delineate which patients have persistent infection were highlighted as important to study. Study results could provide objective data to doctors and patients, and help them understand who might benefit from continued treatment and whether or not the treatment is effective. Special significance was assigned to the study of neuroborreliosis and the unique challenges that it provides in the areas of diagnostic imaging, best regimens for effective treatment, and neural dysregulation. Transmission was an interesting part of our discussion. The word itself can have different meanings in the study of tick-borne diseases. In basic research, it can mean the mechanisms by which the pathogen establishes and maintains infection in its new host. However, several members felt that transmission denotes other ways in which the pathogen might be passed to a host – through potential non-tick mediated transmission (e.g., maternal-fetal, blood supply, sexual transmission) and potential transmission through non-ixodes tick vectors. In any case, the group decided that this large group of research questions would be best left to subsequent reports as they could not be addressed thoroughly in addition to the other topics at hand. At the end of first two meetings, we emerged with the following issues that we believed were important to address during this process. Members were unanimous in their vote for these key issues, although a few members did feel that additional issues should be added. The belief was that these additional issues could be addressed within the broader list as part of the recommendations if the members felt it was appropriate at the time. Table 1: Improving Issues Related to Pathogenesis, Transmission, and Treatment: Complete List of Issues that Could be Addressed in the First Report to Congress Key Theme 1 - Mechanisms of Borrelia burgdorferi Persistence (in Animal Models) Pathogenesis of Bb persistence in animal models Evidence for efficacy of treatment in eliminating Bb in animal models Pathogenesis of Bb infection and persistent infection after antimicrobial treatment in humans Key Theme 2 – Pathogenesis of Continued Signs or Symptoms of Disease Pathophysiology of Lyme disease signs/symptoms, including persistent infection, immune dysfunction, co-infections, neural dysregulation Tools/biomarkers to identify mechanisms of continued signs/symptoms Identification of predictors of disease course Key Theme 3 - Optimal Treatment Regimens Potential treatments that address LD signs/symptoms including persistent infection, immune dysfunction, co-infections, neural dysregulation Efficacy for current treatment regimen for acute, disseminated and chronic signs and symptoms Does infection with >1 pathogen change treatment efficacy Assessing treatment outcome measures Key Theme 4- Transmission of Borrelia burgdorferi Mechanisms by which Bb establishes and maintains infection Evidence for non-tick mediated transmission of Bb Evidence for vectors other than Ixodes ticks transmitting Bb Evidence for effects of Borrelia subspecies on tick transmission rates Once these issues were agreed upon, we narrowed the list to focus on the major questions as a subset to this large list. We have not ranked them in a particular order, and there was not a consensus as to which of the three is most important. While we have noted a single gap that may be important to fill with respect to the priority, this is by no means meant to be exclusive, and for the sake of conciseness we will seek to address any additional gaps we identify in the recommendations themselves. Table 2: Improving Issues Related to Pathogenesis, Transmission, and Treatment: Prioritized List of Issues that Will be Addressed in the First Report to Congress Prioritized Key Issues What mechanisms of B. burgdorferi pathogenesis allow it to persist in some animal species despite a competent immune system and/or antimicrobial therapy? (What are the gaps in human research that need to be addressed to explore this model of pathogenesis in humans?) What is the pathogenesis of persistent symptoms in antibiotic-naïve and antibiotic-treated patients? Are there biomarker(s) to determine the continuing presence of infection? (What are the gaps in research regarding ongoing symptoms related to the effect of delayed diagnosis, immune dysfunction, persistent infection, co-infections and neural dysregulation?) What is/are the best treatment regimens for acute Lyme disease, and for patients with ongoing symptoms who have or have not been previously treated? (GAP: What are the tools needed to measure treatment outcomes in Lyme disease, including but not limited to patient-centered outcomes, clinical practice outcomes, and innovative research tools?) Methods The Chair and Vice-Chair of the Working Group selected two members for each subcommittee to serve as co-chairs (see Table 1). One was a federal employee and the other was a member of the public. For the Pathogenesis, Treatment and Transmission subcommittee, Wendy Adams, MBA, Bay Area Lyme Foundation and a former Lyme patient, and CAPT Estella Jones, DVM, Food and Drug Administration (FDA), were chosen as Co-Chairs. Dr. Jones’ alternate member was originally CDR Tracy MacGill, PhD, from FDA; however, she was subsequently replaced with David Leiby, PhD, also from FDA. A total of approximately 120 persons expressed interest in the Pathogenesis, Treatment, and Transmission subcommittee. These nominees were considered using a two-stage process. The co-chairs reviewed all nominations and identified the nominees who appeared to have substantial experience related to the specific content of the subcommittee. The co-chairs found it easier to have one co-chair complete the evaluation forms at the time of the discussion. The co-chairs then discussed the suitability of the nominees who had substantial tick-borne disease experience. Geographic, medical specialty, and research focus diversity for subcommittee members were considered important to developing the most inclusive and representative report. The mission of the subcommittee required members with scientific, medical/clinical, and advocacy backgrounds. Scientists with deep experience in microbiology and immunology were chosen. Scientists also had either in vitro research backgrounds or had performed numerous animal research studies and could speak to the large body of knowledge related to Borrelia pathogenesis in both antibiotic treated and antibiotic-naïve animal species. Given that the subcommittee is also examining treatment of Lyme and tick-borne diseases, physicians needed to represent a critical part of the subcommittee. The co-chairs sought physicians with expertise in tick-borne disease patient treatment and Lyme disease clinical trial conduct and evaluation. The selected physicians were all co-authors of peer-reviewed studies on tick-borne diseases. The co-chairs chose doctors with board certification in infectious disease, family medicine, neurology, and psychiatry given the diverse, often complicated clinical manifestations that present in Lyme disease patients. When looking for an additional patient advocate to serve on the committee, the co-chairs sought someone with deep experience in Lyme advocacy who was also intimately familiar with Lyme disease outcomes research and the guidelines for treatment from both the Infectious Disease Society of American as well as the International Lyme and Associated Disease Society. While the co-chairs did consider including additional representation from the federal government, the government applicants did not have any tick-borne disease experience that would benefit the subcommittee. Given the substantial expertise of the subcommittee members themselves, the co-chairs determined to tap both expertise within the committee as well as external presenters. The subcommittee was divided into three groups related to the priorities we chose to investigate in the first report to the Working Group, with one member serving on two of the priorities because of the member’s extensive clinical experience. Our first meeting was focused on brainstorming around the subcommittee’s mission – what did each of these focus areas mean? What were the most important aspects of pathogenesis, treatment, and transmission to be covered in the short time frame before our first report was due, and what would need to be covered subsequently? As mentioned in the Issues and Priorities section, different definitions emerged of transmission, for example. Given the diverse viewpoints and expertise of the committee, this time was well spent and helped us focus our activities on areas we thought to be most important to providing recommendations to the Working Group on actions that should be taken to improve the government response to tick borne diseases. Subsequent meetings have been focused on reviewing our priority #1, pathogenesis of persistent disease in antibiotic-naïve or treated animal models. Presentations from inside and outside the Subcommittee were requested and the scientific studies posted and reviewed in advance of the meetings. Presentations were given, with brief Q&A following each presentation, and with time reserved for general discussion about what was learned and how it should be referenced in our final recommendations. Members and meetings of our subcommittee are described in the following tables. Table 3: Members of the Pathogenesis, Transmission, and Treatment Subcommittee: Member Type Stakeholder Group Expertise (Co-chair)Wendy Adams, MBA, Bay Area Lyme Foundation Public Patient Advocate Research grant director, Board Member, Lyme Disease Biobank (Co-chair)Estella Jones, DVM, Food and Drug Administration Federal Public Health Acting Deputy Director, Office of Counterterrorism and Emerging Threats Nicole Baumgarth, DVM, PhD University of California, Davis Public Scientist Immunology; expertise in mammalian infectious disease immunology animal models Patricia K. Coyle, MD, Stony Brook University Public Physician Neurology, Director Multiple Sclerosis Care Center Sam Donta, MD Public Physician Infectious diseases; Lyme physician Brian Fallon, MD, Columbia University Public Physician Neuropsychiatry; clinical trial investigator; Director, Columbia Lyme Disease Center Lorraine Johnson, JD, MBA, Lymedisease.org Public Patient Advocate CEO Lymedisease.org; Principal investigator, MyLymeData David Leiby, PhD, FDA Federal Public Health Chief, Product Review Branch, Center for Biologicals Evaluation and Research (CBER) Elizabeth Maloney, MD Public Physician Family medicine; Medical Director, LymeCME - Tick-borne disease physician education Jon Skare, PhD, Texas A & M University Public Scientist Microbiology; expertise in B. burgdorferi host interactions, complement inhibition Brian Stevenson, PhD, University of Kentucky Public Scientist Microbiology; expertise in B. burgdorferi gene/protein expression during infection Table 4: Overview of Pathogenesis, Treatment, and Transmission Meetings, 2018 Meeting No. Date Present Topics Addressed 1 February 26, 2018 John Aucott, MD, Working Group Chair; Wendy Adams, MBA, Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD Co-Chair Wendy Adams provided and overview on process and logistics; the subcommittee discussed key issues on the preliminary list, clarified key terminologies, revised the existing list of key issues, finalized and voted on the full list of issues. 2 March 5, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Pat K. Coyle, MD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD Two invited presentations; Q/A following the presentations; group discussion on key mechanisms of pathogenesis of infection, effect of Bb on the immune system, variations in antibody response, markers of infection, antigens and antibiotic tests, and seroconversion and infection. 3 March 12, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD Two invited presentations; Q/A following the presentations; group discussion on symptoms of Lyme disease, pathogenesis of B. burgdorferi, roles of metals, gene transfer, and inter-bacterial communication. 4 March 19, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD The group discussed the draft Background Section, and preliminary outline for Priority 1; Sam Donta gave a presentation on the Pathophysiology of Lyme Disease. 5 March 26, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; David Leiby, FDA; Elizabeth Maloney, MD; Jon Skare, PhD The group discussed the process of developing the final report to the working group, as well as the outline for Priority 2. Diego Cadavid gave a presentation on Key Learnings from Animal Models of Borreliosis. 6 April 2, 2018 Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; David Leiby, FDA; Elizabeth Maloney, MD; Jon Skare, PhD The group discussed the status of several items as well as agendas for upcoming meetings. Priority 2 was also discussed, including building out the content of the outline. Priority 3 updated on their group discussions 7 April 9, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD Betty Maloney presented on Evidence based Medicine and GRADE. Betty reported on the evidence for Lyme disease treatment. In addition, Armin Alaedini, PhD presented on Immune Mechanisms in PTLDS. The group discussed his findings on differences in immune profiles of different patient groups 8 April 16, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; David Leiby, PhD; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD Ying Zhang, MD presented on Optimal Antibiotic Regimens for Acute and Persistent Lyme Disease. He compared Lyme disease to tuberculosis in their ability to form persistent cells after antibiotic treatment. Lorraine Johnson, JD, MBA also spoke on Patient Centered Healthcare and MyLymeData. The group discussed the presentations afterward. 9 April 23, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; David Leiby, FDA; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD Brian Fallon reviewed the clinical trial results in Lyme disease. The group also discussed the draft of Priority 2, and discussed and voted on the outline of Priority 3. The group voted on the outline for Priority 3 10 April 25, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; David Leiby, PhD, FDA; James Berger Alternate DFO; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD The group discussed sections of the report and outlined remaining responsibilities that needed to be concluded before finishing the report. The group also voted on the Potential Actions to be included in the report. 11 April 30, 2018 John Aucott, MD Working Group Chair; Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; James Berger Alternate DFO; Nicole Baumgarth, DVM, PhD; Sam Donta, MD; Brian Fallon, MD, MPH; Lorraine Johnson, JD, MBA; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD The subcommittee reviewed and discussed the current version of the Results section for Priorities 2 and 3. They also discussed how to organize the slides. 12 May 2, 2018 Wendy Adams Subcommittee Co-Chair; Estella Jones Subcommittee Co-Chair; Nicole Baumgarth, DVM, PhD; Brian Fallon, MD, MPH; Elizabeth Maloney, MD; Jon Skare, PhD; Brian Stevenson, PhD The group discussed the slide presentation to the Working Group and reviewed logistics and timelines for finishing the report. Table 5: Presenters to the Pathogenesis, Treatment, and Transmission Subcommittee Meeting No. Presenter Topics Discussed 2 Nicole Baumgarth, DVM, PhD, Center for Comparative Medicine, Department of Pathology, Microbiology and Immunology, University of California, Davis Monica E. Embers, PhD, Division of Bacteriology and Parasitology, Tulane National Primate Research Center Borrelia burgdorferi Alters and Evades the Adaptive Immune System Studies of B. burgdorferi Persistence in Animal Models 3 Jon Skare, PhD, Texas A&M Health Science Center, College of Medicine, Department of Microbial Pathogenesis and Immunology, Bryan/College Station, Texas Brian Stevenson, PhD, Professor, Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine Pathogenesis-related features of B. burgdorferi How does B. burgdorferi recognize its environment, and then respond appropriately? 4 Sam Donta, MD, Professor of Medicine (retired), Infectious Disease Pathophysiology of Lyme Disease 5 Diego Cadavid, MD, Vice President, Clinical Development, Fulcrum Therapeutics Key Learnings from Animal Models of Lyme Disease 7 Betty Maloney, MD, President, Partnership for Tick-Borne Diseases Education Armin Alaedini, PhD., Department of Medicine, Columbia University Medical Center Evidence based Medicine and GRADE Immune Mechanisms in PTLDS 8 Ying Zhang, MD, Professor of Microbiology and Infectious Disease, Johns Hopkins Lorraine Johnson, JD, MBA, CEO LymeDisease.org, Principal Investigator, MyLymeData Optimal Antibiotic Regimens for Acute and Persistent Lyme Disease Patient Centered Healthcare and MyLymeData 9 Brian Fallon, MD, Professor of Clinical Psychiatry, Director, Lyme and Tick-Borne Diseases Research Center, Columbia University A Brief Review of the US Clinical Trials Table 6: Votes Taken by the Pathogenesis, Treatment, and Transmission Subcommittee Meeting or Date Motion Results Minority Response 3/2/2018 Approve Issues and Priorities Section of the report for submission to the Working Group Passed 9/0/1/0 No 3/19/2018 Approve the Outline of Priority One section Passed 9/0/0/1 No 3/20/2018 Approve Background Section of the report for submission to Working Group Passed 9/0/0/1 No 4/12/2018 Approve the Outline of Priority Two section Passed 9/0/0/1 No 4/23/2018 Approve the Outline of Priority Three section Passed 9/0/0/1 No 4/30/2018 Approve Priority Three Report Passed 9/0/0/1 No 5/1/2018 Approve Potential actions, Priorities One, Two, Three Passed 9/0/0/1 No 5/4/2018 Approve Final Report Passed9/0/0/1 No Votes were required on the following sections of the report background, methods, issues and priorities, priority one, priority two, priority three, and complete draft of report that is ready to be submitted to the Working Group. Enter the date the vote was initiated e was taken ONLY FOR EMAIL votes. Use the meeting number in Table 2 all other meetings Results Results Section for Priority 1–Mechanisms of Persistence Introduction A hallmark of the Lyme disease-causing bacterium, Borrelia burgdorferi, is its ability to efficiently transmit from feeding Ixodes species ticks to humans and other vertebrates, disseminate throughout the body, and establish long-term, persistent infection. All of this occurs even when a patient has a complete, functional immune system. Very little is known about the mechanisms by which B. burgdorferi accomplishes these tasks. Some spirochete proteins have been identified that play critical roles in establishment of persistent mammalian infection, but almost nothing is known about how they operate. There is strong evidence that B. burgdorferi manipulates its host’s immune system to enable their persistence in reservoir host species. Herein, we focus on key questions that need to be addressed. These include both bacterial and host components that enable persistent infection. Noting that certain antibiotic therapies may not completely clear B. burgdorferi from infected humans or experimental animals, we raise the possibility that the spirochete may use similar mechanisms to avoid killing by host immune system and by antibiotic drugs. Animal models are the most accurate systems to identify bacterial factors necessary for infection, host mechanisms involved with bacterial clearance or tolerance, and to determine whether a therapy can cure infection. We present discussions of the major animal models currently used for studies of B. burgdorferi infection. None of them completely mimic human infection, and advantages and disadvantages of each animal model are presented. Due to space constraints, we opted not to focus on other aspects of B. burgdorferi’s mammal-tick infectious cycle. The mechanisms by which the Lyme spirochete persistently colonizes ticks, why only some species of tick are capable of transmitting B. burgdorferi to humans, and how the bacteria are transmitted from an infected mammal into a feeding tick, are important questions, but of lesser importance to the prevention and clearance of human Lyme disease and have therefore been omitted from this report. (1) Evidence for B. burgdorferi persistence in natural infection Persistence of Borrelia burgdorferi within the reservoir host(s). One of the most pertinent issues with regards to transmission and pathogenesis of Borrelia burgdorferi is to learn the mechanisms by which the bacteria can persistently infect its natural vertebrate reservoirs. Once infected, reservoir species maintain the bacteria for many months, possibly for their entire lives. B. burgdorferi can then transmit into Ixodes ticks via a blood meal as they feed on these environmental reservoir hosts. Thus B. burgdorferi can be transmitted from one mammalian host to another with the help of the tick. There is no strong evidence to suggest that B. burgdorferi can be transmitted from adult to offspring through the eggs (that is, no transovarial transmission) (Radolf, Caimano, Stevenson, & Hu, 2012). In endemic areas of the northeastern and midwestern United States, the predominant vertebrate reservoir species is the white footed mouse, Peromyscus leucopus, but other small mammals and certain bird species can carry B. burgdorferi (Heylen, Müller, Vermeulen, Sprong, & Matthysen, 2015; Richter, Spielman, Komar, & Matuschka, 2000). Grey squirrels are the primary vertebrate reservoir in endemic counties of California (Salkeld et al., 2008). For P. leucopus and other rodents, the infection has no overt pathology associated with it; that is, no arthritis or any other disability is observed (Baum et al., 2012; Schwanz, Voordouw, Brisson, & Ostfeld, 2011). In addition to P. leucopus and birds, other competent reservoirs include dormice, squirrels, and cricetids (Matuschka, Allgöwer, Spielman, & Richter, 1999; Matuschka, Endepols, Richter, & Spielman, 1997; Richter et al., 2000). Note that although B. burgdorferi can cause infection of a number of larger vertebrates, including dogs and humans, many hosts that are significant sources of tick feeding are not considered “competent” reservoirs notably, deer, cattle, and sheep. That is, these species are not necessary to maintain B. burgdorferi in the ecosystem, that is, all are dead-end hosts. However, these animals are critical for maintenance of the tick populations, which has direct impacts on B. burgdorferi and risk of Lyme disease. For example, control of deer herds has been linked to reduction in tick numbers (Rand, Lubelczyk, Holman, Lacombe, & Smith, 2004). Persistence of Borrelia burgdorferi in experimental models of infection; Laboratory mice. In terms of experimental models of Lyme borreliosis, laboratory mice (Mus musculus) have been used widely because they are relatives of the natural reservoir species, they can be readily infected, and some strains of M. musculus display aspects of the pathophysiology observed in human infections (Barthold, 1995; Barthold, Beck, Hansen, Terwilliger, & Moody, 1990; Barthold, de Souza, Janotka, Smith, & Persing, 1993; Barthold, Moody, Terwilliger, Jacoby, & Steere, 1988). The most commonly-used inbred mouse strain for pathology studies is the C3H lineage, which develops B. burgdorferi-induced arthritis more readily than other strains, such as BALB/c or C57BL/6. The genetic basis for the arthritic sensitivity of C3H mice has been associated with variants of several genetic loci (reviewed in (Bramwell, Teuscher, & Weis, 2014)). If the genetic basis for arthritic manifestation is conserved between vertebrates, these studies could be important in identifying factors that link infection with prolonged pathology also in humans. However, no information is yet available to determine whether genetic factors associated with arthritis and other pathologies in mice are also relevant to what occurs in humans. Despite their limitation, notably the absence of erythema migrans (EM) lesions and neurological pathology, mice serve as a critical model to study B. burgdorferi infection and dissemination mechanisms in a mammalian host, as well as to study host immunity to infection, potential mechanisms of persistence and disease tolerance in reservoir species, and Lyme borreliosis. Substantial numbers of reagents are available for immunological and other studies of mice. Such tools are not available for detailed analyses of any other species of experimental animal. Numerous inbred strains of mice have been developed, many of which have defined genetic traits, which facilitates comparisons between animal studies and transfer of cell populations between mice (by contrast, all other model animals are outbred, which complicates animal-to-animal reproducibility). Mice can be readily infected with infected ticks, by transfer of infected tissue, or quantitatively by needle inoculation of cultured spirochetes. The spirochetes disseminate to similar tissues in mice as seen in humans (lymph nodes, joints, heart), and can give rise to some of the pathologies associated with disseminated human disease (for example, lymphadenopathy, arthritis, and carditis). Following infection, experimentally infected mice generate innate and adaptive immune responses to B. burgdorferi (with notable limitations), which control but do not clear the infection, demonstrating the utility of inbred mice as a persistent infection model. The course of B. burgdorferi infection in mice: Experimental infection of mice with B. burgdorferi can be categorized within two phases linked to the murine immune response: an innate phase and an adaptive phase. During the initial innate phase, the spirochetes spread throughout the mouse via the bloodstream, lymphatics, and extracellular matrix (ECM), during which transient splenomegaly and lymphadenopathy are observed (Barthold et al., 1990; Barthold et al., 1993; Tunev et al., 2011). B. burgdorferi has a predilection for connective tissue within the skin, blood vessels, and heart, as well as muscle tissue, and pathologies associated with B. burgdorferi infection of mice is seen at this point (Barthold et al., 1990; Barthold et al., 1993). Several adhesive proteins (adhesins) on B. burgdorferi have been identified that allow them to bind to the host ECM. The adhesins of B. burgdorferi can bind to many host factors, including soluble and insoluble fibronectin, decorin (which coats collagen), integrins, and collagen itself (Brissette & Gaultney, 2014; Coburn, Leong, & Chaconas, 2013; Zambrano, Beklemisheva, Bryksin, Newman, & Cabello, 2004; Zhi et al., 2015). Host glycosoaminoglycan (GAG) structures are also recognized (Fischer, LeBlanc, & Leong, 2006; Leong, Morrissey, Ortega-Barria, Pereira, & Coburn, 1995). Genetic modification of B. burgdorferi that removes adhesins can makes these bacteria less able to infect (Hyde et al., 2011; Seshu et al., 2006; Shi, Xu, McShan, & Liang, 2008; Weening et al., 2008; Zhi et al., 2015). However, others appear unaffected, suggesting that compensatory/backup systems exist to substitute for lost function(s). A complete understanding of the exact residence requirements and the physiologic status of B. burgdorferi in mammalian tissues still is lacking. Increased knowledge about the ways in which B. burgdorferi invades and resides in tissues, however, could provide important insights into novel treatment regimens to eradicate these persistent spirochetes. Perhaps even more striking is the lack of knowledge on the distinct functions for many coordinately regulated gene products of B. burgdorferi, particularly those that are involved in the blood meal transition phase, including outer surface protein C (OspC). Thus, there is a large void in our understanding of the activities for many of the proteins expressed by B. burgdorferi, despite their temporal linkage to initial infectivity and colonization. Enhanced understanding of these early molecular events in borrelial pathogenesis will be critical when attempting to develop therapies that can stop the dissemination and persistence of this pathogen in humans. In the second phase of the host response to B. burgdorferi infection, adaptive immunity develops. Indeed, antibodies are measurable within about one week after infection, beginning in the lymph nodes at the site of initial infection (Tunev et al., 2011). As outlined further below, antibodies of both the IgM and IgG isotypes are produced. In persistently infected mice, a strong and continued production of IgM continues unabatedly and simultaneously with IgG production, which is an unusual immunological phenomenon and may bear on the inability of antibodies to completely clear the infecting B. burgdorferi ((Tunev et al., 2011); Hastey et al, in preparation). Significantly, overall IgM titers decline in infected humans, but there may be a persistence of IgM reactivity for months to years against a small number of proteins in the range of 37-41 and 20-25 kDa. During early infection, IgG antibodies are initially produced against proteins in this range (Aguero-Rosenfeld et al., 1996; Aguero-Rosenfeld, Nowakowski, McKenna, Carbonaro, & Wormser, 1993; Craft, Fischer, Shimamoto, & Steere, 1986; Craft, Grodzicki, & Steere, 1984; Dressler, Whalen, Reinhardt, & Steere, 1993a; Kalish et al., 2001; Karlsson, Mollegard, Stiernstedt, & Wretlind, 1989; Massarotti et al., 1992). A similar pattern is seen in naturally infected dogs and experimentally infected mice (Barthold, 1993; Barthold & Bockenstedt, 1993; Barthold et al., 1993; Barthold, Feng, Bockenstedt, Fikrig, & Feen, 1997). Antibodies are effective in clearing a large number of spirochetes, but not all of the bacteria are eliminated. Passive transfer of serum antibodies from persistently infected mice into naïve mice generally protects against challenge with B. burgdorferi delivered either by tick or syringe inoculation. In contrast, passive antibody transfer does not protect naïve mice from disseminated infection if they are challenged by subdermal implantation of pieces of skin from infected mice containing “host-adapted” spirochetes. This suggests that spirochetes that have established an infection are somehow shielded against host antibodies. Why the antibodies can prevent infections when transferred into another mouse, but cannot clear all of the spirochetes in the host from which they were taken is puzzling. Addressing this question may reveal important mechanisms by which B. burgdorferi evades host immunity. Spirochetes persist within the skin, joints, and ECM components associated with blood vessels (Barthold, Hodzic, Tunev, & Feng, 2006). These B. burgdorferi are associated with collagen fibrils and do not appear to stimulate a robust inflammatory response. Intravital microscope video imaging of fluorescently-tagged B. burgdorferi within mouse tissues indicate that the bacteria are highly motile (M. Wooten, unpublished results). The exact metabolic status of these forms of B. burgdorferi is unknown, as it is difficult to obtain them in numbers commensurate for molecular analyses. Peromyscus leucopus. The white-footed mouse (P. leucopus), is a major wild reservoir of B. burgdorferi in many parts of the U.S. Despite the name, it is distantly related to the domestic mouse, Mus musculus (described above). Because many strains of B. burgdorferi are adapted for optimal infection of P. leucopus, the white-footed mouse serves as an excellent model to identify the bacterial and host components that are necessary for persistent infection. It appears that P. leucopus does not suffer any negative consequences from B. burgdorferi infection, thus is tolerant to the infection. Thus, in its natural hosts, B. burgdorferi seems to manipulate the immune system and avoids triggering significant pathologies. At present, there are limited resources and tools for immunological and molecular studies of P. leucopus, and the animals are relatively difficult and expensive to acquire. Expanding availability of such resources would aid in the study of how B. burgdorferi can successfully subvert host immune responses. Dogs: Dogs can be naturally infected by tick-bite and present to the veterinary clinic with similar disease manifestations as humans, particularly arthritis. Due to the similarities in clinical presentations, they have been used also as a larger experimental animal model. There are conflicting reports as to whether dogs develop EM. In the absence of treatment, young dogs in particular can suffer from multiple bouts of lameness linked to recurring joint inflammation (Appel et al., 1993). Untreated dogs can also be persistently infected (Straubinger, Straubinger, Summers, Jacobson, & Erb, 1998). The advantages of using dogs as experimental model are offset by the ethical considerations and public perception of dogs as pets. Additional drawbacks include the lack of dog-specific immunological tools, and the expense of purchase and maintenance of dogs (for example, ethical treatment of dogs requires large space per animal, regularly scheduled walks, and other demands on time, space, and personnel). Significant large-scale clinical studies on dogs have not been conducted to date, but could provide a currently untapped resource of tissues from infected animals. Rabbits: Rabbits represent another large animal species that has been experimentally infected with B. burgdorferi. In fact it was utilized by Willy Burgdorfer in the initial description of B. burgdorferi (Burgdorfer et al., 1982). Rabbits develop EM lesions with disseminated infection that spreads throughout the skin, lymph node, spleen, liver, joints, eye, and spinal cord tissue, but not the spinal fluid (Foley et al., 1995). Distinct from other animal models, rabbits clear the infection and become protected from reinfection with the same Borrelia strain but they are not protected when another B. burgdorferi strain is used (Shang, Wu, Lovett, Miller, & Blanco, 2001). As such, rabbits do not serve as good model for persistence, but can serve as models of EM development and other aspects of early infection as well as the study of protective immunity. However, the lack of tools for study in rabbits places limitations on the use of this model. Non-human primates: Studies in rhesus macaques and baboons demonstrated that these non-human primates (NHP) present with EM and develop disseminated infection in a manner that is hypothesized to mirror that of humans, due to their genetic relatedness (Hefty et al., 2002; Philipp et al., 1993). For this reason, NHPs are likely the most relevant animal model for Lyme disease. Similar to afflicted humans, NHPs show central nervous system involvement, as well as pathologies associated with peripheral nerves, heart tissue, skeletal muscle, and connective tissue of the joints (England, Bohm, Roberts, & Philipp, 1997; Roberts, Bohm, Cogswell, NorbertLanners, et al., 1995). In many, but not all, instances, cultivation of B. burgdorferi is negative late in infection of NHPs. However, other experimental metrics suggest that they continue to be present in a persistent state. Specifically, B. burgdorferi has been reported as present in both untreated (England et al., 1997; Roberts, Bohm, Cogswell, Lanners, et al., 1995) and antibiotic treated (Crossland, Alvarez, & Embers, 2018; Embers et al., 2012; Embers et al., 2017) animals, based on xenodiagnosis and the detection of both RNA and DNA. (Note: xenodiagnosis is a method by which mammalian infection status is ascertained through the feeding of naïve ticks – acquisition of B. burgdorferi by the feeding ticks indicates that the mammal is infected). More recent studies detected B. burgdorferi in the heart, muscle, and around peripheral nerves in both antibiotic treated and untreated NHPs (Crossland et al., 2018). While the earlier studies in NHP used a more virulent strain of B. burgdorferi, in which strong pathological evidence for disease was presented (Roberts, Bohm, Cogswell, Lanners, et al., 1995), these more recent studies showed overall only very mild pathologies, which are hypothesized to be caused by persistent B. burgdorferi. The lack of clinical manifestations of disease in these NHP must be considered a limitation for this Lyme disease model. In immunosuppressed NHPs, the number of spirochetes in tissues increases substantially (Bai et al., 2004; Cadavid et al., 2004). There are also signs of inflammation observed in cardiac and neural tissues, respectively. However, the relevance of chemically-immunosuppressed NHPs as a model of natural disease in immunocompetent humans is debatable. Furthermore, the limited availability and cost of NHPs, and the ethical considerations of their use, are substantial drawbacks to their use as an infection model. Disease course in humans infected with B. burgdorferi Most individuals who present with classic early symptoms of Lyme disease, for example EM accompanied with flu-like illness, will clear the infection if treated immediately with antibiotics. However, not all infected humans develop an EM inflammatory response, or the EM may not be noticed. The absence of EM creates difficulties in diagnosis, as “flu-like” symptoms alone are not necessarily diagnostic of Lyme disease, and might be ignored by patients or clinicians. Unless treated within the first few weeks of infection, B. burgdorferi-infected patients may develop a wide array of clinical inflammatory conditions involving the nervous system, heart, and/or musculoskeletal tissues. Treatment at this stage can also be successful but may require extended antibiotic treatment. The underlying cause of ongoing disease is unknown and subject of intense discussion. Another subset of patients, including some who initially received antibiotic treatment, develop a persistent condition that is more challenging to treat effectively. These individuals may present with neurologic issues, pain, severe fatigue, and chronic arthritic and/or musculoskeletal sequelae. We lack an understanding of why humans respond differently to the infection. Notably, we lack an understanding as to why some patients develop EM whereas others do not, and why there is such a broad spectrum of pathologies/presentations, as well as why some appear to be less responsive to treatment. A more complete understanding of these differences could inform the development of more patient-specific and distinct treatment regimens. Detailed studies on Lyme borreliosis in humans are limited for obvious reasons. While studies in dogs and NHP can provide surrogates for diseases seen in humans, particularly neuroborreliosis, experimental models using mice are the mainstay in basic science given their ease of use and cost. However, as indicated, there are significant distinctions between human and mouse infection with B. burgdorferi. The most prominent difference is their overall tolerance to infection and absence of disease manifestations, similar to that seen in naturally infected rodent species. This includes the absence of EM lesions, mild-to-absent inflammatory conditions of the heart and joints in most strains, and a general lack of neurological infectivity. The latter might be a result of reduced levels of connective tissue in the brain of small vertebrates relative to larger ones, although this possibility has not been formally studied. Thus, while mice will remain a critical species for studies that reveal the function of B. burgdorferi proteins, the bacteria’s ability to infect and migrate through host tissue, and evade immune responses, there is an absence of robust and cost-effective animal models for the study of other aspects of Lyme disease, particularly neuroborreliosis. Please refer to the discussion below for more details on persistence in humans. (2) Evidence for B. burgdorferi resistance/persistent infection following antibiotic treatment As outlined above, ample evidence demonstrates that B. burgdorferi persistently infects natural reservoir species and experimental animals. Most patients suffering from acute symptoms of B. burgdorferi infection are unable to clear the infection without antibiotic treatment (Steere, 1983). However, the prevalence of subclinical infection of B. burgdorferi in humans is unknown. The concept and findings of persistence of B. burgdorferi following antibiotic treatment is highly controversial. “Persisters”, or dormant variants of bacteria that have not succumbed to therapeutic interventions, have been found for many pathogens including Salmonella typhimurium, Pseudomonas spp. and Mycobacterium tuberculosis, explaining chronic infections with those pathogens despite antibiotic therapies (reviewed in (Lewis, 2010). Both in vitro and in vivo evidence has been presented to suggest that B. burgdorferi may develop persister forms following antibiotic therapy that might cause ongoing signs and symptoms of Lyme disease. It remains to be determined whether borrelial persistence following antibiotic treatment is a genetically encoded characteristic, or is due to environmental conditions such as osmotic shock or inadequate distribution of antibiotics. Thus it remains to be determined whether reports of continued infection of humans or experimental animals after antibiotic therapy were due to insufficient antibiotic dosages or the development of “persister” forms. Studies that distinguish between these potential mechanisms should be considered a high-priority for future research into the mechanisms of Borrelia persistence and the clinical presentations of ongoing signs and symptoms of Lyme disease. Studies from the Lewis (Sharma et al., 2015), Zhang (Feng et al., 2015; Feng, Zhang, Shi, & Zhang, 2016), and Embers (Caskey & Embers, 2015) laboratories have reported the presence of round-body and microcolony forms of B. burgdorferi in stationary cultures following antibiotic treatment. These morphological changes were associated with increased tolerance to antibiotic treatments in vitro. Those studies were precipitated by in vivo reports, first in dogs (Straubinger, 2000; Straubinger, Summers, Chang, & Appel, 1997) and mice (Bockenstedt, Gonzalez, Haberman, & Belperron, 2012a; Bockenstedt, Mao, Hodzic, Barthold, & Fish, 2002; Hodzic et al., 2008; Hodzic, Imai, Feng, & Barthold, 2014; Yrjanainen, Hytonen, Hartiala, Oksi, & Viljanen, 2010; Yrjanainen et al., 2006) and later in NHP (Embers et al., 2012; Embers et al., 2017) of the continued presence of DNA in various tissues of antibiotic (ceftriaxone)-treated animals. In addition to finding spirochete DNA, evidence for antibiotic-resistant B. burgdorferi in vivo included PCR-positivity for RNA, suggesting active infection, as well as positive DNA and immunohistochemistry findings from ticks allowed to feed on antibiotic-treated animals (“xenodiagnosis”) (Bockenstedt et al., 2002; Hodzic et al., 2008), and immunohistochemical evidence of intact spirochetes in tissues. Consistent across all studies, cultures established with tissues from antibiotic treated animals remained negative for B. burgdorferi. It should be noted that in some cases, cultured human specimens were B. burgdorferi positive following antibiotic treatment (Haupl et al., 1993; Liegner et al., 1997; Preac-Mursic et al., 1989). Despite experimental evidence for persistence of Borrelia DNA, RNA and Borrelia-proteins in mice (Bockenstedt et al., 2012a), the interpretation of this data as demonstrating persistence of infection has been challenged. This is because viable spirochetes could not be retrieved from the antibiotic treated host mouse, the tick that fed on the host mouse, and the mice exposed to the ticks. This lack of retrieval has prevented the demonstration of active infection (reviewed in (Shapiro, 2015). While this is accurate, no plausible explanation has been put forward that could account for the persistence and transfer of Borrelia genetic material (DNA) from an infected and antibiotic-treated mouse to a tick that fed on that mouse and the retrieval of the DNA in immunodeficient mice exposed to the DNA-containing ticks. Further work is needed to resolve this issue. One recent study put forward evidence for recrudescent infection of B. burgdorferi in mice that underwent a 30-day treatment regimen with ceftriaxone (Hodzic et al., 2014). That study reported on the resurgence of non-culturable B. burgdorferi in mice at 1-year post-treatment. In that study, PCR analysis for DNA was negative for nearly all tissues for cohorts of mice three and eight months after antibiotic treatment, but was positive for most tissues 12 months after infection. Data were confirmed by xenodiagnoses, demonstrating the presence of DNA and protein in ticks fed upon those mice. Unfortunately, the 12-month time point was the last analyzed. In a recent study, non-human primates were treated for 28 days with doxycycline, four months after their initial infection with B. burgdorferi by tick-transmission. Seven to nine months post-treatment, intact and viable RNA-transcribing spirochetes were recovered by xenodiagnosis and through the use of an in vivo culture system. These organisms were of comparable viability to those recovered from untreated primates (Embers et al., 2017). See the discussion below for details regarding B. burgdorferi in humans following antibiotic treatment. As noted above, the reported experimental treatment failures may be interpreted as evidence that the antibiotics and/or dosages were not adequate, or that persister forms developed in the treated animals. Studies of additional antibiotics, used alone, in combination, or different treatment regimens are warranted, as are further studies into the nature of potential residual spirochetes present in tissues of infected and then antibiotic treated animals. (3) Mechanisms of persistence following natural infection with B. burgdorferi Gene regulatory schemes in ticks as an adaptive response to infect vertebrates The zoonotic lifecycle associated with Lyme disease is very complex. Since the Lyme disease spirochete is not passed through eggs from infected female Ixodes ticks, the larval ticks must acquire B. burgdorferi by feeding on an infected vertebrate (Radolf et al., 2012). Larval acquisition of B. burgdorferi involves unknown chemotactic signals provided by the tick to promote this process (Schwan & Piesman, 2000; Tilly, Rosa, & Stewart, 2008). After molting from larva to nymph, the tick seeks to feed on a new host. This can result in transmission of B. burgdorferi to the new host, which could be a human. For this to occur, the bacteria must survive the blood meal (complement resistance is key here), move to the hemocoel, traverse the hemolymph, and invade the salivary glands while not succumbing to the tick innate immune response (Kazimírová & Štibrániová, 2013; Smith & Pal, 2014; Wikel, 2013). During transmission of B. burgdorferi via the blood meal, Ixodes ticks also secrete immunomodulatory proteins that enable the transmission process (Kazimírová & Štibrániová, 2013; Wikel, 2013). These tick saliva proteins alter both innate and adaptive immune functions by promoting vasodilation and altering platelet aggregation, coagulation, inhibiting complement, skewing neutrophil function, and impairing T and B cell responses (Couvreur et al., 2008; Guo et al., 2009; Juncadella & Anguita, 2009; Kazimírová & Štibrániová, 2013; Kotsyfakis et al., 2006; Ribeiro & Spielman, 1986; Valenzuela, Charlab, Mather, & Ribeiro, 2000; Wikel, 2013). During the blood meal, as B. burgdorferi traffics from the midgut to the salivary glands, dramatic changes in its protein expression profile occur. These enable B. burgdorferi to change from a spirochete adapted to survive in the tick midgut to one that is capable of infection and colonization of a vertebrate. This includes changes in the proteins expressed on the outer membrane of B. burgdorferi. How the spirochete senses the tick feeding is not fully understood, nor are the regulatory pathways known that coordinate repression of tick-specific factors while enhancing production of vertebrate-specific factors. Even though large numbers of proteins are known that are produced coordinately, it is important to emphasize that we know very little about the function of most of these proteins. Given the extent and energy that B. burgdorferi expends to change its surface protein repertoire, it follows that many of the proteins of unknown function are important for borrelial infectivity, colonization, dissemination, and persistence. More detail about the change at the transcriptional and post-transcriptional level is provided below. Dissemination of B. burgdorferi within the vertebrate host After initial infection into the skin, B. burgdorferi divides and then spreads into deeper tissue (Radolf et al., 2012). They are actively motile at this point. They avoid killing by innate immune responses, such as the alternative pathway of complement. There is no evidence to suggest that B. burgdorferi produces any toxins or secretes proteins (for example, proteases) that could break down host tissues (Casjens et al., 2000; Fraser et al., 1997). Instead the spirochete coats itself with host enzymes and other proteins that facilitate dissemination. (See Appendix 1 for more detail). This “hijacking” of host proteins allows B. burgdorferi to invade and inhabit areas that the immune response may not be able to reach easily. Invasion into connective tissues results in further spread of the infection to more distant lymph nodes, joints, cardiac, spleen, and bladder tissues, as well as skin sites. Proteins derived from B. burgdorferi seem to persist within murine connective tissue and may trigger continued inflammation (Bockenstedt, Gonzalez, Haberman, & Belperron, 2012b). Despite these significant advances, we still do not understand much of the molecular details regarding the spread of B. burgdorferi throughout the infected host. The spirochetes spread through the blood stream, lymphatics and/or by movement through solid tissues (Bockenstedt et al., 2012b; Kumar et al., 2015; Moriarty et al., 2008; Tunev et al., 2011). Additional studies are needed to determine the mechanisms underlying the ability of B. burgdorferi to disseminate throughout the vertebrate body. Adaptation to the host environment and immune evasion mechanisms Gene expression and protein production. Pathogens utilize genetic regulatory mechanisms to adapt to the changing environments they encounter during infection to ensure their survival. B. burgdorferi dramatically alters its gene and protein expression profile as it moves between the Ixodes spp. arthropod vector and the mammalian hosts they infect (Radolf et al., 2012; Samuels, 2011). We have only a partial understanding of the regulatory networks that affect borrelial gene expression (Caimano, Eggers, Gonzalez, & Radolf, 2005; Hyde, Shaw, Smith Iii, Trzeciakowski, & Skare, 2009; Jutras, Chenail, et al., 2013b; Jutras, Chenail, Rowland, et al., 2013; Miller, Karna, & Seshu, 2013; Ouyang et al., 2009; Stevenson & Seshu, 2017). Nevertheless, there are still gaps in our knowledge base regarding the schemes that regulate gene expression and protein production in B. burgdorferi. For example, B. burgdorferi encodes only two regulatory schemes defined as two-component regulatory systems (most bacteria have many more than 2 systems). Both systems require soluble regulatory proteins, known as response regulators, and membrane bound histidine kinases. Generally, signals are perceived by the histidine kinase and then transduced to the response regulator via phosphorylation (Stock, Robinson, & Goudreau, 2000). The two response regulators encoded by B. burgdorferi, designated Rrp1 and Rrp2, are required for tick and mammalian (murine) survival, respectively (Boardman et al., 2008; Burtnick et al., 2007; Caimano et al., 2015; Caimano, Eggers, Hazlett, & Radolf, 2004; Caimano et al., 2007; He et al., 2011; Kostick et al., 2011; Rogers, Terekhova, et al., 2009; Yang, Alani, & Norgard, 2003). However, the signal that is perceived in either system is not known. This is admittedly a specific example, but this type of difference/distinction applies to other borrelial regulatory pathways as well. Numerous other regulatory proteins have been identified in B. burgdorferi, several of which function independently of either Rrp1 or Rrp2 (ref. Stevenson & Seshu, 2018, in press). With this paucity of information comes the need to further characterize these processes. In addition to conventional transcriptional regulation, the presence of small, non-coding RNAs, or sRNAs, have been identified in nearly every bacterium examined, including B. burgdorferi (Adams et al., 2017; Arnold et al., 2016; Popitsch, Bilusic, Rescheneder, Schroeder, & Lybecker, 2017). Recent data indicates that B. burgdorferi make over a thousand different sRNA molecules (Arnold et al., 2016; Popitsch et al., 2017). Regulation mediated by sRNA may occur by binding to transcripts, for example, messenger RNA or mRNA, which are translated into protein. The binding of sRNA to specific mRNA occurs via specific sequence recognition and either enhances or inhibits translation to proteins and, as such, represents a post-transcriptional form of regulation. Other sRNAs may bind and sequester regulatory proteins, thereby preventing the proteins from interacting with other target sites. How sRNAs fit into the pathogenic potential of B. burgdorferi is not well understood but it is clear that some are produced specifically during mammalian infection (Adams et al., 2017) and are needed for optimal infectivity. B. burgdorferi also produces at least three distinct RNA-binding proteins, BpuR, SpoVG, and CsrA. Both BpuR and SpoVG bind to their own mRNAs and control their own translation (Jutras, Chenail, et al., 2013a; Jutras, Chenail, Rowland, et al., 2013; Savage et al., 2018). They also bind specifically to other mRNAs, and appear to control translation of numerous proteins. Some RNA targets of CsrA have been identified, including mRNAs for flagellar proteins, and csrA mutants dysregulate levels of numerous transcripts and proteins, and are defective in mammalian infection (Karna, Prabhu, Lin, Miller, & Seshu, 2013; Karna et al., 2011; Sanjuan, Esteve-Gassent, Maruskova, & Seshu, 2009; Sze & Li, 2011; Sze et al., 2011). Despite our lack of knowledge about the regulatory mechanisms operative in B. burgdorferi, several genes that are required for tick and vertebrate adaptation have been identified. B. burgdorferi efficiently responds to signals that quickly convert the spirochete from the tick-specific gene expression pattern into the compendium of gene expression and protein production required for vertebrate host colonization. A number of the signals likely to be in play here (for example, temperature, pH, O2 and CO2 levels, bacterial replication rates, and nutrients) have been analyzed in vitro (Arnold et al., 2016; Carroll, Cordova, & Garon, 2000; Carroll, Garon, & Schwan, 1999; Hubner et al., 2001; Hyde, Trzeciakowski, & Skare, 2007; Ojaimi et al., 2003; Schwan, Piesman, Golde, Dolan, & Rosa, 1995; Seshu, Boylan, Gherardini, & Skare, 2004; Stevenson, Schwan, & Rosa, 1995; Yang et al., 2000)(Stevenson and Seshu, 2018, in press) and, in most instances, mirror what is likely to occur following infection given the concordance of proteins produced during this process. However, there are mammalian-specific signals that have not been recapitulated in vitro, specifically the robust repression of ospA (Akins, Bourell, Caimano, Norgard, & Radolf, 1998; Brooks, Hefty, Jolliff, & Akins, 2003; Iyer et al., 2015). Recently, a comparison of total transcript profiles were compared between in vitro grown B. burgdorferi, infected and fed larval ticks, infected and fed nymph ticks, and host adapted B. burgdorferi by temporary surgical implantation of sterile dialysis membranes that encase B. burgdorferi into the peritoneal cavity of rats (Iyer et al., 2015), with the latter treatment intended to approximate what occurs during mammalian infection. While one might predict that the tick-associated transcript profiles would be similar, the fed larvae and nymphs were vastly different and looked significantly different than in vitro mammalian grown B. burgdorferi. Most striking was the differences observed with the in vivo adapted profile. Several genes that were highly expressed in the tick or in culture were turned off in the mammalian host adapted state. Many other genes are shut down in fed larvae only and those are highly expressed during mammalian infection (Iyer et al., 2015). From this analysis it is clear that B. burgdorferi is capable of significantly altering its transcriptional repertoire. These changes are undoubtedly critical for transmission, survival within the tick, and for the establishment and maintenance of mammalian infection. Studies that address these events at the molecular level need to be continued and expanded upon. Among other outcomes, such studies can reveal new targets for improved therapies to prevent and cure human infection. Antigenic variation. The antigenically variable surface protein VlsE is essential for persistent infection of vertebrates. B. burgdorferi strain B31 that do not contain the linear plasmid that encodes vlsE, or strains that are missing the expression site for vlsE, can briefly infect mice but are quickly and completely cleared coincident with antibody development (Bankhead & Chaconas, 2007b; Labandeira-Rey, Seshu, & Skare, 2003). Infectious B. burgdorferi possess a series of unexpressed, silent “vls cassettes” adjacent to the vlsE expression gene. During mammalian infection, vlsE undergoes repeated recombination events of silent vls cassette fragments into multiple sites of the vlsE gene, via a gene conversion mechanism (Zhang, Hardham, Barbour, & Norris, 1997; Zhang & Norris, 1998a). This system can yield up to 1032 VlsE variants. This level of variability means that each B. burgdorferi cell can carry a distinct form of VlsE that may not be recognized by the antibodies made against it, particularly late in infection (Zhang, Hardham, et al., 1997; Zhang & Norris, 1998a, 1998b). The crystal structure of VlsE showed that the variable domains, which are subjected to the recombination events described, map to the surface exposed face of VlsE (Eicken et al., 2002). A much less immunogenic part of the VlsE protein, that does not undergo antigenic changes, is IR6 or C6. While this antigen is often used in the serological evaluation of patients for its stable expression, the antigen maps to an internal structure within VlsE (Eicken et al., 2002), that is much less easily accessible to the immune system and thus predicted to generate much less strong antibody responses. Variation of vlsE occurs only during vertebrate infection, never during tick colonization or laboratory cultivation (Zhang & Norris, 1998b). The mechanisms that control the ability to vlsE to undergo variation at only the critical time of mammalian infection remain to be elucidated fully. While the recombinase RuvA was identified as critical for recombination (Dresser, Hardy, & Chaconas, 2009; Lin et al., 2009) other regulators remain to be identified. No “host adapted” strategy has recapitulated the recombination that ensues in vivo. Production of the VlsE protein, and transcription of its gene, is tightly regulated by B. burgdorferi: it is not produced during tick colonization, but is induced during transmission from tick to mammal (Bykowski et al., 2006). Because of its importance in persistence, identifying and targeting the signal(s) required for host specific recombination in vlsE, and for activation of VlsE production, would provide important insights into how this critical B. burgdorferi factor enables evasion of persistent infection, and could be new targets for protective or curative treatments. Resistance to Complement Killing. A cascade of interacting proteins (“the complement cascade”) exists in the blood of all vertebrate species, which that can cause lysis of invading bacteria. The complement cascade is key in the rapid, innate host defense, by detecting and clearing foreign invaders. B. burgdorferi is known to resist all three pathways of complement cascade activation (classical, lectin, and alternative). For B. burgdorferi, complement resistance is also important for its ability to survive the blood meal of the feeding tick. Without complement resistance mechanisms at this stage of their lifecycle, B. burgdorferi would not be capable of transmission to mammals that they infect. Several Borrelia proteins mediate resistance to the alternative pathway of complement activation. Specifically CspA (also known as CRASP-1), CspZ (CRASP-2), and several Erp proteins, notably ErpP (CRASP-3), ErpC (CRASP-4), ErpA (CRASP-5), and OspE (Alitalo et al., 2002; Alitalo et al., 2001; de Taeye, Kreuk, van Dam, Hovius, & Schuijt, 2013; Hallström et al., 2013; Hammerschmidt et al., 2012; Hellwage et al., 2001; Hovis et al., 2006; Kraiczy, 2016b; Kraiczy, Hartmann, et al., 2004; Kraiczy, Hellwage, et al., 2004; Kraiczy & Stevenson, 2013; McDowell et al., 2003; Rogers, Abdunnur, McDowell, & Marconi, 2009; Rogers & Marconi, 2007; van Dam et al., 1997). All of the aforementioned borrelial proteins bind to the complement component factor H or factor H-like protein 1, which prevent activation of the alternative pathway of complement. B. burgdorferi strains that cannot bind factor H binding in vitro, as with B. burgdorferi cspA mutants (Hartmann et al., 2006; Kenedy, Vuppala, Siegel, Kraiczy, & Akins, 2009; Kraiczy, Hellwage, et al., 2004), do not impede the complement activation cascade, resulting in the deposition of the so-called “terminal membrane attack complex” onto the surface of those B. burgdorferi, which results in its death. Such killing does not require antibodies or any other factor. For that reason, B. burgdorferi mechanisms of factor H-acquisition may be important targets for developing new antibacterials. C4BP binding to both B. burgdorferi and Borrelia recurrentis, the agent of louse-borne relapsing fever, has been reported but in the case of B. burgdorferi, the identified protein has not been characterized at the molecular level (Grosskinsky et al., 2010). C4BP is another soluble regulator in the complex complement scheme, and it affects a step common to the classical and lectin pathways of complement activation. Binding of C4BP to the surface of pathogens alters the stability of the classical convertase that is required for complement activation. In practice, the binding of factor H and C4BP together could render a foreign system resistant to all complement-dependent killing, which, in theory, could be mediated by only two encoded proteins. However, extracellular pathogens have evolved numerous back up strategies that ensure that the loss of any one gene does not put the organism at a selective disadvantage. The numerous borrelial factor H binding proteins illustrate this point. By analogy with other extracellular pathogens (for example, Staphylococcus aureus) that have many redundant complement neutralizing activities, it is likely that B. burgdorferi utilizes a similar strategies with heretofore unidentified proteins that prevent innate clearance, as well as antibody-mediated clearance (see below). Recently, an inhibitor that is specific for the classical pathway of complement activation was identified in B. burgdorferi (Garcia, Zhi, Wager, Höök, & Skare, 2016). The classical pathway, in general, works by engaging the C1 complement complex with antibody-bound antigen. The interaction of C1 with the antigen-antibody complex results in complement activation and killing of a targeted cell. Somewhat surprisingly, the B. burgdorferi generated inhibitor mapped to the carboxy-terminal domain of the fibronectin binding protein adhesion, BBK32. BBK32 is a bipartite molecule with a structurally unordered amino terminal half that attains a stable structure when it binds to fibronectin (Kim et al., 2004). The carboxy (C-)-terminal half is highly ordered but had no known function. Overlay binding analyses demonstrated that BBK32 could recognize the classical complement C1 complex, composed of C1q, C1r, and C1s proteins (Garcia, Zhi, Wager, Höök, et al., 2016). Subsequent characterization showed that the C-terminal BBK32 fragment bound to the initiating protease in the classical pathway, C1r, and strongly inhibited activation of the classical pathway in vitro (Garcia, Zhi, Wager, Höök, et al., 2016). Thus, it is possible that BBK32 may serve to minimize complement-dependent, antibody-dependent killing of B. burgdorferi and, if so, could contribute to persistence in the face of the humoral-based adaptive immune response. Alterations to Innate Immune Response Activation and Quality As discussed above, upon its entry into the mammalian host, B. burgdorferi is perceived by the innate immune response. However, tick salivary proteins play a role in suppressing the host immune system as long as the tick vector is attached. Most of the information we have about these processes come from experimental infection in mice. Numerous isogenic gene knockout strains of C57BL/6 mice have been useful in delineating the role of various innate immune pathways in the context of B. burgdorferi infection. B. burgdorferi engenders a strong innate immune response by engaging toll-like receptors (TLR)2, TLR5, TLR7, TLR8, and TLR9 (Cervantes et al., 2011; Cervantes et al., 2013; Dennis et al., 2009; Marre, Petnicki-Ocwieja, DeFrancesco, Darcy, & Hu, 2010; Parthasarathy & Philipp, 2018; Petzke, Brooks, Krupna, Mordue, & Schwartz, 2009; Wooten et al., 2002). All receptors utilize the downstream MyD88 adaptor protein to induce pro-inflammatory cytokines, and in the case of TLR7, TLR8, and TLR9 induction via nucleic acids, they may also induce Type I interferons (IFN) (Cervantes et al., 2011; Cervantes et al., 2013; Petzke et al., 2009). Predominantly, infection with B. burgdorferi is sensed by interaction of lipid moieties from their lipoprotein constituents with TLR2/1 heterodimers (Fikrig et al., 2009; Hirschfeld et al., 1999; Salazar et al., 2009; Shin et al., 2008; Wooten et al., 2002). Although there is some debate in regard to how TLR7, 8, or 9 activate Type I IFN following B. burgdorferi infection, and whether other innate receptors are involved, given that IFN-induction was found even in the absence of the adaptor-molecules MyD88 and TRIF (Hastey, Ochoa, Olsen, Barthold, & Baumgarth, 2014), it is clear that Type I IFN stimulated genes are induced (Cervantes et al., 2011; Hastey et al., 2014; Miller et al., 2013; Petzke et al., 2009). The induction of Type I IFN is surprising, given that the known protective functions of these cytokines are usually associated with viral and intracellular bacterial infection. Type I IFN induces a so-called “antiviral state”, rendering cells less susceptible from viral invasion, and they drive adaptive immunity towards a strongly inflammatory TH1-type immune response, by inducing IL-12 production by dendritic cells. Further work is required to determine whether the induction of Type I IFN helps the control of infection, or whether it might be driving some observed immune pathology (Miller, Ma, Bian, et al., 2008) and possible persistent infection, as seen with other infections (reviewed in (McNab, Mayer-Barber, Sher, Wack, & O'Garra, 2015). Interpretation of studies conducted in vitro are complicated by the fact that culture-grown B. burgdorferi expresses lipoproteins rapidly lost by B. burgdorferi in the mammalian host, thus in vitro studies of cellular activation with culture-grown Borrelia may not relate to in vivo events. However, the fact that mice lacking the innate adaptor molecule MyD88 show much higher Borrelia-loads than mice expressing this protein (Bolz et al., 2004; Liu, Montgomery, Barthold, & Bockenstedt, 2004), demonstrates the importance of innate immune activation in the control of B. burgdorferi levels in tissues. Alterations to the Adaptive Immune Response Infection of immunodeficient SCID mice (Barthold, Sidman, & Smith, 1992; Hodzic, Feng, Freet, & Barthold, 2003; Schaible et al., 1990; Schaible, Kramer, Museteanu, et al., 1989; Zimmer et al., 1990) or B6-Rag1 null mice (McKisic & Barthold, 2000; McKisic, Redmond, & Barthold, 2000), which lack T and B cells, and thus a functional adaptive immune system, results in progressive disease, demonstrating a role of the adaptive immune system in disease prevention. However, given that even fully immunocompetent mice cannot clear the infection, the data suggest that while the adaptive immune system is activated, it is not fully functional. The mechanisms underlying the ultimate ineffectiveness of the immune response are not understood, but are likely multifaceted (reviewed in (Tracy & Baumgarth, 2017)). As discussed above, serum transfer studies with serum from infected mice have demonstrated that antibodies can protect from a challenge infection, even though the antibodies are unable to clear the infection in the host from which they were derived. Another puzzling aspect of the antibody response is that the protective capacity of the serum seems initially to increase with ongoing infection, i.e., serum transferred from mice at day 30 of infection was less protective than serum transferred from a mouse infected for 60 days. However, by about 3-4 months after infection, this protective capacity reversed, such that sera from mice infected for about 1 year were less protective than those taken from day 60 of infection. Although it is currently not possible to measure all antibodies generated against B. burgdorferi, the studies suggested that this was not due to a lack of antibody production, but rather a sign that the generated antibodies no longer provided protection (Barthold & Bockenstedt, 1993). These studies suggest dramatic changes in the functionality of the developing antibodies. The mechanism underlying these change is unknown, but likely important for the understanding of immune protection and immune evasion by B. burgdorferi. Similar to SCID and RAG-/- mice, B cell deficient (B6-Igh6 null) mice cannot effectively control B. burgdorferi tissue loads and disease progression, whereas infection of T cell deficient (B6-Tcr b//d null) mice develop a disease course that parallels fully immunocompetent mice, with disease remission (McKisic & Barthold, 2000; McKisic et al., 2000). Disease resistance and “tolerance” mechanisms are likely outcomes of evolutionary adaptation. Initial studies with various gene-targeted mice provided evidence that B cells control Bb-infection and disease, while a/b or g/d T cells had limited effects (Fikrig et al., 1996; Keane-Myers & Nickell, 1995; Lim, England, DuChateau, Glowacki, & Schell, 1995; McKisic & Barthold, 2000). Indeed, adoptive transfer of naïve T cells into infected B6-Rag1 null mice exacerbates disease, whereas transfer of B cells induces disease remission. Immune sera from T cell-deficient mice, but not B cell-deficient mice, were shown to be protective against challenge (McKisic et al., 2000) and to induce disease remission (Barthold et al., 2006). Infection of T cell deficient mice stimulates the production of Bb-reactive IgM, and IgG3, and lesser amounts of IgG1 and IgG2b (McKisic et al., 2000). One of the few, but notable differences of T cell deficient mice compared to wild type controls is a significant reduction in class switch recombination from IgM to IgG, suggesting that T cells do participate in the B cell response to B. burgdorferi. B. burgdorferi induces strong B cell responses in lymph nodes in both immunocompetent and T cell-deficient mice. Patients with early Lyme disease have increased levels of both B. burgdorferi specific and non-specific IgM in serum and a high level of constitutively activated B cells (Sigal, Steere, & Dwyer, 1988; Steere, Hardin, Ruddy, Mummaw, & Malawista, 1979). This is followed by rising levels of total serum IgG, including antibody to an expanding array of B. burgdorferi proteins (Aguero-Rosenfeld et al., 1996; Aguero-Rosenfeld et al., 1993; Dressler, Whalen, Reinhardt, & Steere, 1993b; Kalish et al., 2001). Experimentally infected mice also have a significant rise in both specific and non-specific IgM and IgG, as well as an increase in peripheral lymph node B cell numbers (Yang et al., 1992). While these earlier studies described some of the induced antibody responses as non-specific, as discussed B. burgdorferi selectively expresses a number of proteins in vivo that are unrelated to the antigens used for serology (Akins et al., 1995; Suk et al., 1995; Wallich, Brenner, Kramer, & Simon, 1995), thus it cannot be concluded that there is strong non-specific component to the antibody response. Indeed, experimental studies in mice suggested the induction of mostly Borrelia-specific antibodies (Tunev et al., 2011). As infection proceeds in humans, IgM titers decline, but there is a curious persistence of IgM reactivity for months to years against a small number of proteins in the range of 37-41 and 20-25 kDa on immunoblots made from B. burgdorferi lysates, as discussed above. Antibiotic-treated humans (Nowakowski et al., 1997; Pfister et al., 1986; Weber et al., 1986) and mice (Piesman et al., 1997), which have recovered from infection, are susceptible to re-infection, suggesting suboptimal or no immunologic memory. Indeed, recent studies in mice showed a lack of memory B cell and long-lived plasma cell induction following B. burgdorferi infection that correlated with a rapid collapse of germinal center responses, usually responsible for immunological memory (Elsner et al., 2015). Thus, the data provide strong evidence for immune evasion mechanisms that reduce the induction and/or maintenance of effective T-dependent B cell responses (Elsner et al., 2015; Hastey et al., 2012). This reduction in immune response effectiveness was seen also when B. burgdorferi-infected mice were vaccinated with a non-relevant antigen (influenza), providing evidence for a more generalized alteration of the immune system during B. burgdorferi infections. The mechanisms Borrelia employs to suppress effective long-term immunity remain unknown and this constitutes a significant gap in our understanding of Borrelia-pathogenesis and disease. The discussed changes in B. burgdorferi protein expression that occur during infection are likely to also reduce the effectiveness of the adaptive immune response. At least some proteins that trigger strong antibody responses are down-regulated during the chronic phase of infection (Liang, Nelson, & Fikrig, 2002). OspC, a T-dependent antigen that is essential for initial colonization of mammalian hosts is rapidly down-regulated during mammalian infection (Gilmore, Kappel, Dolan, Burkot, & Johnson, 1996; Grimm et al., 2004; Mbow, Gilmore, & Titus, 1999; Tilly et al., 2006). However, when induced to be expressed, OspC can trigger a strong and effective antibody response leading to antibody-mediated clearance (Embers, Alvarez, Ooms, & Philipp, 2008; Liang, Jacobs, Bowers, & Philipp, 2002). Antigenic variation is another process that likely reduces effectiveness of antibodies against B. burgdorferi. As outlined above in detail, the variable surface antigen E (vlsE) locus is a major immunodominant surface protein of B. burgdorferi that undergoes extensive and rapid antigenic variation in mammalian hosts (reviewed in (Norris, 2014)). vlsE recombination is critical for B. burgdorferi persistence and the ability of B. burgdorferi to reinfect a host following antibiotic treatment (Bankhead & Chaconas, 2007a, 2007b; Lawrenz, Wooten, & Norris, 2004; Rogovskyy & Bankhead, 2013; Rogovskyy et al., 2015). The lack of vlsE variation was shown to result in the rapid clearance of B. burgdorferi. Recently, mathematical models have been put forward to suggest that a strongly immunodominant variable surface protein such as VlsE may prolong immune responses long enough to drive immune exhaustion (Johnson, Kochin, Ahmed, & Antia, 2012), an intriguing idea that requires testing in the context of B. burgdorferi infection. Possible Survival Strategies of B. burgdorferi Occupation of specific sites in the mammalian body. Analyses of infected mammals shows a predilection of B. burgdorferi for cartilaginous tissues, such as joints. The physical structures of such tissues may impair distribution of antibodies and/or sentinel immune cells, either of which could contribute to bacterial persistence in immunocompetent individuals. The nature of those host tissues may also impede distribution of antibiotics during treatment. VlsE and other proteins as protective shields. The VlsE protein forms an elongated structure, with a spreading domain at the end away from the bacterial surface (it may be said to resemble a tree, with a narrow trunk and spreading foliage up high). The variable regions of VlsE are in the distal “foliage” region. It is possible that VlsE contributes to B. burgdorferi persistence by shielding other proteins that are below the VlsE “canopy”. Another possibility is that some of the B. burgdorferi outer surface proteins that can attach to host proteins and sugar moieties enable use of those host factors as camouflage. That could both block access by antibodies and mis-identify the bacteria as “self” to the host’s immune system. Neither of these hypotheses have been examined in detail. Round body/cyst formation. Formation of altered forms of B. burgdorferi, known as “cysts” or “round bodies”, have been observed when B. burgdorferi cells were exposed to stressful culture conditions (Alban, Johnson, & Nelson, 2000; Brorson & Brorson, 1997, 1998; Brorson et al., 2009). However, there is significant concern that the forms observed under in vitro environments are not relevant to mammalian infection. Recently, round body derivatives were observed in feeding nymphal ticks (Dunham-Ems, Caimano, Eggers, & Radolf, 2012). The absence of the regulatory protein RpoS resulted in a higher frequency of round body formation, but this distinct morphotype was seen even for the wild type parent strain, albeit at lower frequency (Dunham-Ems et al., 2012), and may be due to differences in bacterial water-control pumping mechanisms. Interestingly, these rounded, enlarged forms of B. burgdorferi could be quickly revert back to their normal morphology by placement in rich media (Dunham-Ems et al., 2012). As noted above, it is not known whether the formation of round bodies is a genetically-encoded phenomenon, or a physical response to changing osmotic conditions and whether it occurs at other points in the spirochete’s life cycle. Studies to assess whether these forms can be visualized during experimental mammalian infection may provide important insight into this unique adaptive form. If they exist, detection of alternate forms, coupled with reversion to normally shaped spirochetes, would suggest that B. burgdorferi exploit this mechanism to “hide out” during periods of stress, for example, oxidative stressors, nutritional limitation, or antimicrobial activity, until conditions improve. Evidence for intracellular residence An important aspect of persistent infection is the ability of the pathogen to initially evade complete sterilization by innate immunity and then to survive the subsequent adaptive immune response. In the case of B. burgdorferi infection, the host mounts a strong, but as discussed above, not fully functional humoral immune response. However, despite these responses, the spirochete is able to persist. B. burgdorferi is categorized as an extracellular pathogen. Several in vitro studies have shown that these spirochetes may invade and survive within a variety of cultured mammalian cells, including primary fibroblasts and endothelial cells (Dorward, Fischer, & Brooks, 1997; Klempner, Noring, & Rogers, 1993; Livengood & Gilmore, 2006a; Ma, Sturrock, & Weis, 1991; Wu, Weening, Faske, Höök, & Skare, 2011). These observations led to a hypothesis that B. burgdorferi might invade host cells as an intracellular niche to hide out and evade the humoral immune response. Whether or not such an event occurs in vivo is not known and provides another important gap in our knowledge that has potential implications for the treatment of patients, since not all antibiotics effectively cross cell membranes. (5) Clinical evidence of persistence/immune evasion Is there evidence for intracellular localization in human studies? The tissue models, discussed above, which showed some invasion of B. burgdorferi into a variety of cell types, were followed up with two human studies that suggested intracellular B. burgdorferi sequestration (Aberer, Kersten, Klade, Poitschek, & Jurecka, 1996; Miklossy et al., 2008), one in macrophages and keratinocytes from culture positive human skin biopsies and the other within astrocytes. However, these studies did not provide definitive proof due to study limitations, including an absence of important controls and/or the use of non-specific staining. Evidence of persistent B. burgdorferi in humans The finding of B. burgdorferi persistence in animal models also extends to human disease. While detailed and extensive searches for direct evidence of B. burgdorferi persistence has not been undertaken, multiple case reports and small studies, using a variety of detection methods, document persistent infection in humans. The gold standard is culture. Other direct detection methods include detection of B. burgdorferi DNA or RNA by PCR, detection of intact spirochetes in tissue by immunofluorescent labeling, or detection of protein fragments of B. burgdorferi. As it is with other infectious agents, the potential that positive culture or PCR results reflect inadvertent laboratory contamination must be carefully considered (Fallon et al., 2008; Marques et al., 2014). Evidence of persistent B. burgdorferi in antibiotic-naïve humans B. burgdorferi can be difficult to culture yet positive cultures from patients with long-standing, untreated disease have been documented in the literature (Aberer et al., 1996; Levy et al., 2012; Oksi et al., 1996; Preac-Mursic et al., 1993; Reimers et al., 1993). PCR evidence of B. burgdorferi persistence in untreated disease is more extensive, both in terms of numbers and types of specimens ADDIN EN.CITE.DATA (Brzonova, Wollenberg, & Prinz, 2002; Feder, Abeles, Bernstein, Whitaker-Worth, & Grant-Kels, 2006; Hidri et al., 2012; Karch & Huppertz, 1993; Karma, Pirttila, Viljanen, Lahde, & Raitta, 1993; Karma et al., 1996; Kempf et al., 2015; Leslie et al., 1994; Leverkus, Finner, Pokrywka, Franke, & Gollnick, 2008; Levy et al., 2012; Maimone et al., 1997; Matera et al., 2014; Mikkila, Seppala, Viljanen, Peltomaa, & Karma, 2000; Muellegger et al., 1996; Muller, Itin, Buchner, & Rufli, 1994; Nocton et al., 1996; Nocton et al., 1994; Oksi et al., 1996; Oksi et al., 2007; Paparone, 1995; Rigot et al., 2015; Stinco et al., 2014; Zamponi et al., 1999). Microscopy has also been used to document the presence of B. burgdorferi (Aberer et al., 1996; Levy et al., 2012; Oksi et al., 1996; Preac-Mursic et al., 1993). Evidence of persistent B. burgdorferi in humans with prior antibiotic treatment for Lyme Disease Published cases of culture-proven treatment failure have been relatively rare. However, they do exist despite the lack of available culture tests for clinical use. This has been most commonly reported from skin samples (Hunfeld, Ruzic-Sabljic, Norris, Kraiczy, & Strle, 2005; Pfister et al., 1991; Preac-Mursic et al., 1989; Strle, Maraspin, Lotric-Furlan, Ruzic-Sabljic, & Cimperman, 1996; Strle et al., 1993; Weber, Wilske, Preac-Mursic, & Thurmayr, 1993), but also from cerebrospinal fluid (Liegner et al., 1997; Pfister et al., 1991; Preac-Mursic et al., 1989), blood (Oksi et al., 1999; Viljanen et al., 1992), and ligamentous tissue (Haupl et al., 1993). Notable were the reports by Hunfeld (Hunfeld et al., 2005; Hunfeld, Ruzic-Sabljic, Norris, Kraiczy, & Strle, 2006) in which B. burgdorferi was cultured from the site of a prior EM rash in 19 (1.7%) of 1148 skin culture positive antibiotic-treated EM cases. Five of these 19 cases were studied in great depth; none reported a second tick bite, all took their medication as prescribed (amoxicillin for 14 days (1), ceftriaxone for 14 days (1), cefuroxime for 14 days (2), or azithromycin for 6 days (1)). Culture results from four of the five patients revealed the same genospecies before and after treatment, suggesting these were persistent infections rather than reinfections. Reinfection in endemic areas is not rare. Thus, symptoms or signs that emerge months to years later may be due to re-infection rather than the recrudescence of an old infection and culture, PCR and microscopy cannot distinguish between the two. Therefore, in these instances, supplemental clinical information regarding the nature of the signs and symptoms could be informative. For example, individuals presenting with another episode of an EM lesion following antibiotic treatment are likely to have a repeat infection (Nadelman et al., 2012). Numerous case reports have been published demonstrating PCR evidence of persistent B. burgdorferi DNA after antibiotics therapy in humans treated for Lyme disease (Battafarano, Combs, Enzenauer, & Fitzpatrick, 1993; Bradley, Johnson, & Goodman, 1994; Cassarino, Quezado, Ghatak, & Duray, 2003; Chancellor, McGinnis, Shenot, Kiilholma, & Hirsch, 1993; Coyle et al., 1995; Fraser, Kong, & Miller, 1992; Frey et al., 1998; Kantor, 1994; Lawrence, Lipton, Lowy, & Coyle, 1995; Liegner, 1997; Maiwald, Stockinger, Hassler, von Knebel Doeberitz, & Sonntag, 1995; Mikkila et al., 2000; Nocton et al., 1996; Nocton et al., 1994; Oksi et al., 1999; Oksi et al., 2007; Tager et al., 2001). Positive PCR results in the immediate post treatment could represent debris from dead bacteria that has yet to be cleared. However, with the exception of joints, where bacterial clearance can be substantially delayed and positive PCR results for synovial fluid or tissue samples may not reflect current infection (Li et al., 2011), the clearance of dead bacteria occurs within days to weeks of killing (Lazarus, McCarter, Neifer-Sadhwani, & Wooten, 2012). It should be noted that in the majority of the papers cited in the text and the appendix, the specimens that produced positive PCR results were obtained 6 or more months following antibiotic treatments, making delayed clearance a less likely explanation. The NIH human xenodiagnosis study found PCR evidence of B. burgdorferi in the tick lysate culture obtained from a patient with persistent symptoms of Lyme disease following prior antibiotic treatment that was administered more than a year before the xenodiagnostic specimen was obtained (Marques et al., 2014). Antigenic components of B. burgdorferi have been reported after antibiotic treatment using an assay that detects OspA antigen in the cerebrospinal fluid after dissociation from complexed B. burgdorferi antibodies (Lawrence et al., 1995). In this case report, OspA and PCR positivity were detected in the cerebrospinal fluid of a patient with seronegative neurologic Lyme disease on four occasions after separate courses of antibiotic treatment for relapsing-remitting symptoms. As noted above, the available laboratory tools for demonstrating the presence or absence of viable B. burgdorferi in humans have significant limitations. In light of the human case evidence discussed above, the in vitro appearance of persisters in antibiotic treated cultures of B. burgdorferi (Feng, Shi, et al., 2016; Feng et al., 2014; Sharma et al., 2015) (an accepted bacterial status in other infections) and given the ability to tightly control the timing of infections in experimental animals, these models remain critical to the study of potential persistent B. burgdorferi infections (Fallon et al., 2008; Lawrence et al., 1995). Summary B. burgdorferi utilizes many mechanisms to infect a mammalian host and then to evade the immune system and establish persistent infection, either in antibiotic-naïve or antibiotic treated hosts. Some B. burgdorferi research has been carried out on cultured bacteria. Culture studies are easier to control for exogenous factors and they are also less expensive. However, the relationship between findings in culture and in mammalian host are unknown, given the considerable changes B. burgdorferi undergoes as it changes its environment, for example when it moves from vertebrate to invertebrate host and vice versa. Several different animal models have contributed substantially to our knowledge of the bacteria’s ability to evade the mammalian immune system and to survive antibiotic treatment. Inhibition of the complement system and suppression of strong inflammatory responses are examples of effective evasion of innate immunity by B. burgdorferi. Strategies for antigenic variation by B. burgdorferi, and the suppression of effective B cell responses contribute to a lack of bacterial clearance by the adaptive immune system as discussed here. Potential Actions Promote research on models of B. burgdorferi infection and the mechanisms of disease processes in humans with an emphasis on pathologies that are currently lacking, for example, neuroborreliosis Research on the mechanisms inducing human disease processes induced by B. burgdorferi infection has been sparse and should be prioritized in the future. Each animal model has unique advantages and disadvantages for understanding human disease and Borrelia persistence. Studies using these models can contribute to a deeper understanding of immune evasion and disease processes as well as host-pathogen interactions and the biology of this pathogen. However, none of the existing animal models has fully captured the clinical picture of humans suffering from the long-term consequences of neuroborreliosis and the development of such a model is needed. Another as of yet incompletely understood mechanism, is how B. burgdorferi (and other bacterial species) survive cultures containing antibiotics at concentrations and establish metabolically inactive persister cells. These in vitro identified “persister” bacteria and existing evidence for their potential presence in vivo in some animals and humans treated with antibiotics should be further examined in animal model and human studies to determine whether their continued presence may contribute to, or be responsible for ongoing clinical signs and symptoms of Lyme disease in humans. Further studies on mechanisms of B. burgdorferi survival during infection processes and its tolerance to antibiotics and other stresses As shown in mice, B. burgdorferi seem to hamper the production of high-quality long lasting antibodies, which can control B. burgdorferi infection levels, but cannot clear the infection. The lack of long-lasting responses may also explain that reinfections are common in endemic areas. Furthermore, some evidence has been provided recently to suggest that the inhibition of strong adaptive immunity during B. burgdorferi infection extents to responses to other pathogens, such as influenza. Whether this more general immune suppression occurs in humans should be further studied, as it may have diagnostic and treatment implications for patients who are simultaneously infected with more than one tick borne pathogen. B burgdorferi uses complex mechanisms to survive in immunocompetent mammals, including humans, seemingly even in the presence of antibiotic treatment. More studies are urgently needed to learn how to overcome mechanisms of Borrelia persistence and its relationship to human Lyme disease. Results Section for Priority 2–What is the pathogenesis of persistent symptoms in antibiotic-naïve and antibiotic-treated patients? Are there biomarker(s) to determine the continuing presence of infection? (What are the gaps in research regarding ongoing symptoms related to the effect of delayed diagnosis, immune dysfunction, persistent infection, co-infections and neural dysregulation? Lyme disease is a multi-system, multi-staged infection with diverse symptomology. Patients may present with a simple rash or with a multitude of symptoms and signs in either inpatient or outpatient settings. This section discusses what is currently known about the pathophysiology of Lyme disease and areas where additional knowledge is required to reduce diagnostic delays and improve patient care. Acute Lyme disease The most common sign of human infection with B. burgdorferi is the erythema migrans (EM) lesion. In a trial that was well-designed to characterize presenting manifestations of Lyme disease, 78% of the 183 subjects who were symptomatic and classified with definite Lyme disease had an EM rash (Steere & Sikand, 2003). However, other sources have reported lower rates of EM rashes ("Lyme Disease," 2017; Schwartz, Hinckley, Mead, Hook, & Kugeler, 2017). EM lesions are typically annular in shape. The majority of EM lesions are solid-colored; in the Lyme vaccine trial, the classic “bulls-eye” pattern, which exhibits central clearing, was present in only 9% (Smith et al., 2002)). In the untreated, the rash will expand in size for days to weeks before clearing. To satisfy the CDC surveillance case criteria for the rash, primary EM lesions must be ≥ 5cm in size (approximately 2(CSTE, 2017)). Figure 1 highlights the varied appearance of EM lesions. Publicly available information from health care providers as well as scientific literature often incorrectly identifies the “bulls-eye” lesion as synonymous with EM instead of noting that it is a relatively uncommon EM pattern. Such errors are potentially confusing to patients and clinicians. The reasons for different rash presentations in patients are not known. Recent studies in non-human primates, however, showed that despite being infected with an identical strain, only 3 of 10 infected rhesus macaques developed erythema at the site of infection, and only 1 of the 3 could be classified as a bona fide EM lesion. Like humans, the non-human primates used in the studies were genetically heterogeneous (Embers et al., 2017); therefore, human genetic heterogeneity may be a factor in the variable appearance of EM lesions. The EM lesion is a manifestation (sign) of the localized immune response to B. burgdorferi in the skin at the bite. Recognition of the bacteria by the immune system leads to the recruitment of inflammatory cells and, ultimately, redness of the overlying skin. The inflammatory response continues and follows along as B. burgdorferi disseminates through the skin, which explains the expanding nature of the rash. (See Figures 1a through 1f below for examples of EM rashes or go to CDC Lyme Disease) Image Figure 1a. “Classic” Lyme disease rash: circular red rash with central clearing that slowly expands Image Figure 1b. Expanding rash with central crust: red. expanding lesion with central crust Image Figure 1c. Multiple rashes, early disseminated Lyme disease Image Figure 1d. Red, oval-shaped plaque on trunk Image Figure 1e. Circular red rash with central clearing that slowly expands Image Figure 1f. Bluish rash without central clearing Image Figure 1g. Red-blue lesion with central clearing on back of knee In addition to the EM lesion, other manifestations of acute Lyme disease include many non-specific “flu-like” symptoms which are similar to those of other acute infectious diseases. The most common early manifestations of Lyme disease (see Figure 2) include arthralgia (joint pain), headache, myalgia (muscle pain), fatigue, and fever. It’s important to note that the number and severity of these different disease manifestations can vary greatly from patient to patient and that some infected patients may be asymptomatic during this stage of their infection. In many Lyme-endemic states in the US, the seasonal cycle of tick encounters is used to help doctors distinguish Lyme disease from other infectious diseases that produce similar nonspecific symptoms such as influenza and other viral infections. The majority of Lyme disease infections result from nymphal tick bites. In the Northeast and Midwest, the seasonal peak of nymphal tick populations occurs in the late spring and early summer, when influenza and other viruses are not circulating in adult populations. However, in other areas where B. burgdorferi-infected ticks reside (West Coast, Southern states), ticks interact with and infect humans year round (Clark, Oliver, McKechnie, & Williams, 1998; Goddard, Embers, Hojgaard, & Piesman, 2015; Salkeld et al., 2014; Sanders & Oliver, 1995). In California, for example, 32% of patients with EM and confirmed Lyme disease reported to the California Department of Public Health in 2014 were reported between October and April (CDPH, 2014). The lack of tick seasonality introduces another challenge to arriving at the correct diagnosis. This is especially true in early infection, when two-tiered serologic testing for Lyme disease is less than 50% sensitive (Molins et al., 2014) and the EM lesion may or may not be present. Image Figure 2. Common manifestations and conditions of different stages of Lyme disease. Early acute (3 days to weeks after infection): flu-like symptoms and EM (70-80%). Early disseminated (weeks to months after infection): multiple EM, carditis, meningitis, facial nerve palsy, and radiculoneuropathy. Late disseminated (more than 3 months after infection): arthritis, peripheral neuropathy, and autonomic neuropathy. In patients with systemic symptoms at the time of the EM rash, macrophage-derived IL-1β and TNF-α are present (Mullegger et al., 2000). In a subsequent study of 27 early acute patients, the 5 patients whose T cells produced IFN-γ usually had no symptoms whereas the 22 patients whose T cells did not product IFN-γ had an average of 4 symptoms. Convalescent cytokine production, however, did not correlate with initial symptom types, duration of symptoms prior to treatment, or clinical evidence of spirochetal dissemination (Glickstein et al., 2003). In humans, B. burgdorferi disseminates widely via hematogenous, lymphatic and tissue routes. Common dissemination sites include the musculoskeletal system, skin, the nervous system, and heart (Coyle & Schutzer, 2002). The multi-systemic nature of the infection and the diversity of specific host-pathogen interactions results in a wide spectrum of pathology (Duray & Steere, 1988). Patients with continuing signs and symptoms after initial antibiotic treatment Patients, whether they are antibiotic-naïve or have received prior antibiotic treatment for Lyme disease, can have a wide array of ongoing symptoms and signs (see Table 7). The most prominent symptoms are fatigue, musculoskeletal pain, neurocognitive dysfunction, and sleep disturbances, but also include a number of other symptoms such as headache, paresthesias, and palpitations. A recently published paper identified 25 signs and symptoms in which the severity of each manifestation was significantly greater in patients with well-defined PTLDS than healthy controls (Rebman et al., 2017). The differences between the two groups was especially striking for these nine symptoms: fatigue, joint pain, difficulty focusing/concentrating, muscle pain, memory impairment, word finding, sleep disturbance, neck pain, and irritability. Image Figure 3. Patients with CLD reported the ten following symptoms as severe or very severe: fatigue (48.3%), sleep impairment (40.8%), joint pain (39.1%), muscle aches (36.1%), other pain (34.4%), depression (33.8%), cognitive impairment (32.3%), neuropathy (31.6%), headaches (22.7%), and heart-related issues (9.6%). (DOI: 10.7717/peerj.322/fig-2) The underlying pathophysiology in patients who have persistent symptoms associated with Lyme disease, whether having had some antibiotic treatment or no prior antibiotic treatment, is poorly understood. Pathologic studies in humans are limited. Although there are tissue-specific differences, in general, perivascular infiltrates of lymphocytes, macrophages and plasma cells are prominent. Tissue necrosis is uncommon. Early in the course of Lyme disease, a number of inflammatory correlates have been identified that could explain some of the general symptomatology (Soloski et al., 2014). And in some patients following currently recommended courses of treatment for early Lyme disease, similar inflammatory markers can be found to persist for months after therapy (Soloski et al., 2014). Recent studies have begun to identify biological markers associated with persistent symptoms to a greater extent than in comparison to healthy or recovered controls, including increased expression of interferon alpha (Jacek et al., 2013), IL-23 (Strle, Stupica, Drouin, Steere, & Strle, 2014), the chemokine CCL19 (Aucott et al., 2016), CRP (Uhde et al., 2016), and antineuronal antibodies (Chandra et al., 2010; Jacek et al., 2013). Biomarkers that present early in infection in some studies have been identified as predictors of the development of later PTLDS include endothelial cell growth factor and IL-23 (Strle et al., 2014) and CCL-19 (Aucott et al., 2016). Research studies of human tissue (surgical tissue, biopsy, post-mortem tissue) are critical to advancing the scientific understanding regarding the pathophysiology of this infection and how diagnostic and treatment paradigms might be changed to more appropriately care for patients with Lyme disease. From the perspective of the causative organism, B. burgdorferi, the only putative virulence factor identified thus far appears to be surface proteins, including many surface-exposed lipoproteins, but even so, the mechanism(s) by which they induce specific symptomatology needs further definition. If the lipoprotein affects host neurologic or other metabolic functions, this may support the association of this potential virulence factor with clinical symptoms. Data from the mouse model, using a related organism, Borrelia turicatae, indicates that widespread cerebral inflammation (ie, microgliosis) occurs in mice persistently infected with B. turicatae that cannot be explained by the presence of spirochetes in the brain parenchyma (Gelderblom et al., 2007). Subsequent studies with B.turicatae showed that borrelial lipoproteins can cross the blood brain barrier from the periphery to cause brain inflammation (Londono & Cadavid, 2010); these were not intact Borrelia spirochetes but lipoproteins perhaps from liposomes detached from whole cells or “blebs”. Brain inflammation was demonstrated by upregulation of genes in the brain tissue that are known to be upregulated in acute brain infection. These studies demonstrate that systemic bacterial infections can release proinflammatory products that cross the blood-brain-barrier to induce brain inflammation. Translational questions that emerge from this research include whether Borrelia burgdorferi similarly releases lipoproteins into the circulation that cross the blood brain barrier and induce central nervous system inflammation and thereby affect brain metabolism and cerebral blood flow, abnormalities of which have been demonstrated in prior neuroimaging studies of patients with Lyme disease (Donta, 2012; Fallon et al., 2009; Logigian et al., 1997) or might lipoproteins (and/or yet to be identified virulence factors) have a direct effect on neural tissue. Supporting a potential role for these lipoproteins in human central nervous system Lyme disease, prior research has identified OspA antigens in the cerebrospinal fluid (Coyle et al., 1995; Schulze, Jordan, Williams, & Dolan, 2017; Schutzer et al., 1997). The pursuit of further research with B. burgdorferi lipoproteins and with possible other virulence factors should result in information that would be of both basic and applied value in the management of patients with persistent Lyme disease. Lyme Arthritis In Lyme arthritis, patients have intermittent intervals of joint pain and swelling. The knee is the most commonly involved joint; wrist, shoulder, ankle and elbow involvement is also common. Smaller joints are infrequently affected. Pathologic findings include synovial tissue infiltrates comprised of macrophages, T cells, B cells, and plasma cells which results in villous hypertrophy, vascular proliferation, and fibrin deposition (Duray & Steere, 1988). The latter is not universal but may be prominent in some. Three autoantibodies have been identified that are associated with antibiotic refractory Lyme arthritis (antibodies against endothelial cell growth factor (ECGF), apolipoprotein B-100, and matrix metalloproteinase 10); in one study from this research lab, the levels of anti-ECGF auto-antibodies correlated with obliterative microvascular lesions in synovial tissue – supporting a role in pathogenesis (Londono et al., 2014). Table 7. Signs and Symptoms of Late Lyme Disease* Category Signs and Symptoms Central Nervous System Headache, Stiff neck, Difficulty thinking/concentrating, Delayed processing time, Memory loss, Sleep disturbances, Word searching, Extreme irritability, Sensory hyperarousal (photo, sound, smell, taste), Weakness, Seizures, Tremors, Gait abnormalities, Poor balance, Stroke syndromes Peripheral Nervous System-Autonomic Facial nerve palsy; Altered sensation in trigeminal nerve; Spinal or radicular pain; Burning, stabbing, aching, or shock sensations; Numbness or tingling; Muscle weakness, atrophy, fasciculations; Dizziness on standing Neuropsychiatric Depression; Anxiety; OCD, psychosis (rarely) Musculoskeletal System Joint pain/swelling; Myalgia; Muscle cramps/spasms; Tendonitis; Overuse syndromes Cardiac Dyspnea; Shortness of breath; Nonproductive cough; Dizziness; Syncope; Palpitations; Rhythm disturbances on ECG (eg, AV blocks, SA node dysfunction, atrial fibrillation) Optic Diplopia; Blurred vision; Pain in/around eyes; Change in color vision; Flashing lights; Peripheral waves/phantom images Constitutional Symptoms Fatigue; Fever Gastrointestinal and Genito-urinary Systems Diarrhea/constipations; Urge incontinence; Irritable bladder/interstitial cystitis * The table is not meant to be inclusive of all symptoms. Lyme Carditis Acute symptomatic cardiac involvement, which occurs in 4-10 percent of patients (Krause & Bockenstedt, 2013) most frequently results in various degrees of atrio-ventricular block, including complete heart block that can result in sudden death (Forrester et al., 2014). Symptoms of acute myocarditis include dyspnea, palpitations, and chest pain. Autopsy specimens from four cases of sudden death demonstrated perivascular interstitial infiltrates comprised of lymphocytes, plasma cells and macrophages (Muehlenbachs et al., 2016). Scattered foci of necrotic cardiomyocytes were seen in one. The conduction system was examined in two; both had inflammatory infiltrates involving the AV node, in one case it was necrotizing. In the other, the inflammation also involved the sinoatrial node. In later disease, the usual cardiac manifestations are those of palpitations and chest pain, without the occurrence of complete heart block. The potential link between Lyme disease and dilated cardiomyopathy is controversial. A study of 110 patients with unexplained dilated cardiomyopathy, demonstrated that 20% had a cardiac biopsy which was PCR positive for Borrelia burgdorferi sensu lato species (Kuchynka et al., 2015) and an earlier case report documented the isolation of Borrelia burgdorferi from the myocardium of a man with long-standing cardiomyopathy (Stanek, Klein, Bittner, & Glogar, 1990). Lyme Neuroborreliosis Neurologic involvement in Lyme disease can occur as part of the early dissemination or later in the infection. During early dissemination the most commonly recognized neurologic syndromes are facial nerve palsy (although other cranial nerves can be affected), meningitis, and acute painful radiculoneuritis (Bannworth’s syndrome). The facial nerve palsy may be bilateral and accompanied by a multi-symptom complex, as well as EM. Meningitis typically presents with headache and stiff neck. However, some patients without headaches may have meningitis. These patients may manifest with radicular pains, later showing weakness in the extremities or facial nerves and will have CSF markers that are typical for Lyme meningitis. Occasionally patients present with a chronic meningitis, that is, persisting/worsening symptoms over 4 weeks. Patients can also present with an acute encephalitis (producing an abnormal level of consciousness, sleepiness, mood swings, psychosis, confusion, delerium, or behavioral changes), acute cerebellitis (cerebellar syndrome), and acute transverse myelitis. Acute painful radiculoneuritis can present with spine pain and dermatomal/myotomal features, but typically without headache or stiff neck. There may be simultaneous EM and/or facial nerve weakness. There is an age specific syndrome, affecting children and adolescents, involving intracranial hypertension. However, intracranial hypertension as a manifestation of neurologic Lyme Disease can occur in adults as well (Nord & Karter, 2003). Patients present with headache and have papilledema on fundoscopic examination. Although intracranial hypertension has been associated with obesity, Lyme patients who present with this manifestation are not typically obese. Most neurologic conditions due to late Lyme disease may be categorized as peripheral neuropathy, encephalopathy or encephalomyelitis; the latter is quite rare in the US. Individual presentations of late neurologic disease are often complex and may include cranial neuritis (not exclusive to CN VII, any of the cranial nerves may be involved), motor and sensory neuropathies, upper motor neuron weakness or ALS-like disorders, MS-like disorders, autonomic dysfunction, movement disorders, cerebellar syndromes, seizures, neuropsychiatric disease and dementia (Arav-Boger, Crawford, Steere, & Halsey, 2002; Bloom, Wyckoff, Meissner, & Steere, 1998; Coyle & Schutzer, 1991, 2002; Fallon & Nields, 1994; Halperin, 1995; Halperin, Kaplan, et al., 1990; Halperin, Krupp, Golightly, & Volkman, 1990; Halperin et al., 1988; Harvey & Martz, 2007; Logigian, Kaplan, & Steere, 1990; MacDonald, 1988; Reik, 1993). CDC surveillance case data demonstrate that 10-15% of Lyme disease patients develop neurologic Lyme disease (Schwartz et al., 2017). However, the surveillance case data may not capture some clinical cases because the surveillance case definition requires patients to be positive on two-tier testing; patients with neurologic disease may not meet this laboratory criterion (Logigian et al., 1999). Although neurologic manifestations of Lyme disease are not rare, the diagnosis can elude clinicians (Pachner, 1988). Diagnostic delay may be attributed to the long latent period experienced by some patients (Coyle & Schutzer, 2002; Logigian et al., 1990), making it difficult to connect current symptoms to a distant tick bite; in many cases there is no history of a recalled bite (Berger, 1989). In a 1990 study of chronic neurologic Lyme disease, Logigian et al. found that chronic peripheral and chronic central nervous system complaints developed 16 and 26 months, respectively, post-rash (Logigian et al., 1990). Delayed diagnosis may also reflect the challenges posed when neurologic Lyme disease does not present with the triad of cranial neuritis, radiculitis or meningitis. For example, in a CSF study of multiple erythema migrans (Strle, Ruzic-Sabljic, Cimperman, Lotric-Furlan, & Maraspin, 2006), 9 of 10 cases of culture-positive Borrelia afzelli infection in the CSF were not detected clinically, most likely because the manifestations were dizziness, cognitive problems, and headaches. Clinician under-appreciation of potential neurologic complications due to Lyme disease and over-reliance on serologic testing or testing too early in the infection prior to antibody formation may also be factors. Patients may complain of headache, neck stiffness, pain, numbness, weakness, memory or concentration difficulties, gait disturbances, clumsiness, bladder or bowel dysfunction, hyperacusis, hearing loss (Logigian et al., 1990), seizures, and visual loss, amongst others ADDIN EN.CITE.DATA (Arav-Boger et al., 2002; Bloom et al., 1998; Coyle & Schutzer, 1991, 2002; Dattwyler, Halperin, Pass, & Luft, 1987; Dattwyler, Halperin, Volkman, & Luft, 1988; Fallon et al., 2008; Fallon & Nields, 1994; Halperin, 1995; Halperin, Kaplan, et al., 1990; Halperin, Krupp, et al., 1990; Halperin, Little, Coyle, & Dattwyler, 1987; Halperin et al., 1988; Harvey & Martz, 2006; Logigian et al., 1997; Logigian et al., 1990, 1999; Reik, 1993). See Table 7 for additional symptoms. Additionally, neurologic symptoms are often associated with nonspecific symptoms (Coyle & Schutzer, 1991, 2002; Dattwyler et al., 1987; Dattwyler et al., 1988; Halperin et al., 1987; Logigian et al., 1990, 1999). The most common manifestation of neurologic involvement in late Lyme disease is cognitive impairment, which in more severe cases is categorized as encephalopathy. The cognitive impairment is typically subtle to mild. Dementia, however can be a rare manifestation of neurologic Lyme disease. In a series of 1594 European patients with dementia, 1.25% had evidence of intrathecal B. burgdorferi antibody production in the CSF; 7 of these 20 patients improved or stabilized after antibiotic therapy (Blanc et al., 2014). Whether and how B. burgdorferi is able to establish persistent localization in the nervous system is uncertain. In vitro experiments with three different neural cell lines demonstrated that B. burgdorferi could invade and remain viable in these cells (Livengood & Gilmore, 2006b). If intraneuronal persistence occurs in humans, it would be unlikely that host defenses would attack and destroy such cells. Furthermore, an intracellular location would protect the bacteria against some antibiotics (Donta, 2002). (See P 3 for additional discussion) Neuropsychiatric manifestations Neuropsychiatric symptoms may emerge at various stages of Lyme disease. Neuropsychiatric symptoms include emotional, behavioral, sensory, and cognitive dysfunction. The primary focus for this section will be on disturbances of mood. Studies in adults (Aucott, Rebman, et al., 2013) and children (Fallon et al., 1994) suggest that depressive symptoms are not elevated in patients who present with early erythema migrans. If Lyme disease is not detected until later in infection however, mood disorders are common. Various well-documented case reports document that psychiatric symptoms in rare cases may be the predominant feature of Borrelia burgdorferi infection, such as OCD (Pachner, 1988), psychosis (Hess et al., 1999), mania (Pasareanu, Mygland, & Kristensen, 2012), Tourette’s Syndrome (Riedel, Straube, Schwarz, Wilske, & Muller, 1998), and panic/depersonalization (Fallon & Nields, 1994). In each of these cases, psychiatric treatment was initially less effective than anticipated but antibiotic therapy led to marked improvement or resolution, highlighting the importance of cerebrospinal fluid studies in atypical neuropsychiatric cases. Three of these cases (Hess et al., 1999; Pasareanu et al., 2012; Riedel et al., 1998) met criteria for Lyme encephalitis based on the presence of a lymphocytosis in the CSF as well as B. burgdorferi-specific antibody production. Cross-sectional studies of persistently symptomatic antibiotic-treated Lyme disease reveal that irritability and depressive symptoms are common. In a study of adult patients with chronic neurologic Lyme disease (half of whom who had had early antibiotic treatment), extreme irritability was noted in 26% and depression in 37%. In a large study conducted out of an academic center’s rheumatology clinic, patients with persistent symptoms after well-documented Lyme disease had a nine-fold greater rate of current depression than patients whose Lyme-related symptoms had resolved (45.2% vs 5%) ((Hassett et al., 2008). In a recent report of patients with post-treatment Lyme disease (Doshi et al., 2018), suicidal thoughts were reported by one in five patients, largely restricted to those who had concurrent depression; while this rate was not greater than in a comparison group of those with HIV infection, it was 4 times greater than in the non-patient control group. Because active suicidal intent was reported by 12.5% of those with suicidal thoughts (2 of 16), this report highlights the importance of screening for depression and suicidality in patients with chronic symptoms, a finding consistent with an earlier case series (Bransfield, 2017). Similar to the cross sectional reports on depression, cross sectional studies of adults with signs and symptoms following initial antibiotic treatment for Lyme disease revealed similar results in terms of anxiety. In one study, patients with persistent symptoms (n=31) had a 2-fold greater rate of a current anxiety disorder than recovered patients (n=40) (29% vs 15%) (Hassett et al., 2008). Anxiety and mood disturbances, as noted earlier, may reflect the stressors associated with chronic illness for which both antibiotic and non-antibiotic treatments have been insufficiently helpful. Pathophysiologic Mechanisms The pathophysiology of neuropsychiatric symptoms in Lyme disease is multi-factorial, involving both physical and psychologic processes. The physical processes may reflect the inflammatory response to active infection, damage from the prior inflammatory reaction, or possibly molecular mimicry induced by the infection leading to cross-reactive antibodies which target neural tissue (Fallon, Levin, Schweitzer, & Hardesty, 2010). The intrapsychic, interpersonal, and societal stressors that accompany Lyme disease may lead to psychological symptoms, particularly alterations in mood such as depression and anxiety. These stressors include persistent pain, cognitive impairment, severe fatigue, financial and job losses, functional disability, interpersonal isolation, invalidation by medical professionals, altered sense of identity due to inability to live up to one’s own expectations as a caregiver or financially self-supporting adult, or sense of a foreshortened future. Mechanisms of disease that may underlie neuropsychiatric symptoms are many: Inflammation. B. burgdorferi infection in the CNS or elsewhere in the body leads to the production of inflammatory mediators. Inflammation – whether due to active infection or past infection – has been associated with several neuropsychiatric disorders (Miller & Raison, 2016) and with central nervous system Lyme disease (Fallon et al., 2010). Confirmation that inflammation itself is causing neuropsychiatric symptoms is challenging as inflammation is characterized by an interplay between pro- and anti-inflammatory cytokines, some cytokines have dual functions, cytokines affect microenvironments in the body that may not be readily detected by serum or CSF studies, and there is a cascade of interactions elicited by the cytokines themselves that have down-stream effects that may then be the most proximal cause of the neuropsychiatric symptoms. Studies of the CSF of patients with neurologic Lyme disease have shown increased levels of the proinflammatory cytokines IL-6, IL-8, IL-12, IL-18 and interferon γ and of the chemokines CXCL12 and CXCL13 ((Grusell, Widhe, & Ekerfelt, 2002; Weller, Stevens, Sommer, Wietholter, & Dichgans, 1991; Widhe et al., 2002; Widhe et al., 2005). In one study, the magnitude of IL-6 in human serum and CSF correlated with disease activity in neurologic Lyme disease (Weller et al., 1991). As noted earlier, altered cytokine and chemokine levels have been shown in the serum of patients with continuing signs and symptoms after initial treatment of Lyme disease. Because specific outer surface proteins of B. burgdorferi are active inducers of inflammatory cytokines (Weis, Ma, & Erdile, 1994) and because certain cytokines themselves, when peripherally administered, can induce prominent mood disorders (Miller and Raison 2016), it is reasonable to review how cytokines might induce neuropsychiatric symptoms. Peripherally circulating cytokines can traverse the blood brain barrier by active transport, by passive transport across leaky sections of the blood-brain barrier, or possibly through lymphatic vessels. A recent article identified several ways in which cytokines may lead to neuropsychiatric symptoms, described below (Rosenblat & McIntyre, 2017). Altered Monoamine levels. Proinflammatory cytokines (eg. TNF-α, IL6) can alter monoamine levels, possibly by increasing the production of enzymes that enhance the metabolism of tryptophan and by enhancing cytokine-dependent breakdown of 5-HT. Depletion of tryptophan and of 5-HT can induce depression and impair cognitive function. Pro-inflammatory signaling can also decrease the levels of dopamine and norepinephrine. Inflammatory cytokines influence glutamate metabolism by acting on microglia and astrocytes, leading to a net increase in glutamate and overactive glutamate receptors, which then enhances excitotoxicity and causes impaired neuroplasticity. When cytokines increase the activity of the microglial cells, the microglia (the macrophages of the CNS) which normally prune unused neural circuits may then aberrantly prune neural circuits that are dedicated to mood and cognitive function. A destructive feedback loop then develops in which activated microglia themselves produce more cytokines, leading to more inflammation and aberrant pruning. The overactivated microglia and excess proinflammatory cytokine production may then also contribute to altered monoamine and glutamate levels, as well as to the production of reactive oxygen species and consequent oxidative stress. In healthy individuals, there is a balance between reactive oxygen species (ROS) and anti-oxidants. Certain affective disorders, such as bipolar disorder, have been shown to have increased ROS and decreased antioxidants that neutralize the ROS; this causes neurodegeneration in key areas that subserve cognition and mood (reviewed in (Rosenblat & McIntyre, 2017)). Proinflammatory cytokines, such as IFN, TFN, and IL6, up-regulate activity of the hypothalamic-pituitary-adrenal axis and increase systemic cortisol levels. When inflammation is chronic, hypercortisolemia occurs which is associated with a range of negative sequelae, such as problems with cognition, mood and physical symptoms. The neuromodulator quinolinic acid was shown in one study to be elevated in the CSF of patients with neurologic Lyme disease (Halperin & Heyes, 1992). Quinolinic acid is an excitotoxin and N-methyl-Daspartate (NMDA) agonist; this study showed higher levels in those with elevated white blood cells in the CSF than in those with Lyme encephalopathy. Because quinolinic acid is a NMDA agonist and therefore affects a receptor known to be involved in learning, memory, and synaptic plasticity, alterations of neuromodulators need to be considered as potential contributors to the neurologic and cognitive deficits seen in many Lyme disease patients. The overall impact of inflammation is to decrease neuroplasticity in the brain which may then lead to neuropsychiatric symptoms. Altered neural function. Brain imaging studies have demonstrated altered blood flow and metabolism among patients with persistent cognitive problems after Lyme disease and among patients with persistent symptoms ((Logigian et al., 1997); (Fallon, Keilp, Prohovnik, Heertum, & Mann, 2003; Fallon et al., 2009). The host response to infection with B. burgdorferi is presumed to underlie these blood and metabolism abnormalities in well-defined cases of Lyme encephalopathy, as prior studies have demonstrated improvement in cerebral blood flow after antibiotic therapy (Donta, Noto, & Vento, 2012; Logigian et al., 1997). Studies in which both blood flow and metabolism were studied simultaneously showed comparable regions in the brain of decreased metabolism and flow; this close coupling of flow and metabolism suggests that the primary deficit is not one of blood flow but one of metabolism (Fallon et al., 2009). However, the vasculature can be directly compromised, as confirmed by European cases of CNS vasculitis due to neuroborreliosis (Keil, Baron, Kaiser, & Deuschl, 1997; Oksi et al., 1996; Topakian, Stieglbauer, & Aichner, 2007). Autoantibodies and Molecular mimicry. Autoantibodies may also play a role in neurologic and neuropsychiatric symptoms during or after B. burgdorferi infection. As a result of B. burgdorferi infection, autoreactive antibodies may develop to myelin and myelin components ((Garcia-Monco, Coleman, & Benach, 1988; Martin, Martens, Sticht-Groh, Dorries, & Kruger, 1988; Suchanek, Kristoferitsch, Stanek, & Bernheimer, 1986). B. burgdorferi's surface glycolipids may elicit cross-reactiveantibodies (Garcia-Monco & Benach, 1997). Other studies demonstrate cross-reactive polyclonal and monoclonal antibodies which recognize flagellar antigenic determinants as well as epitopes on neural cells ((Aberer et al., 1989; Fikrig, Magnarelli, Chen, Anderson, & Flavell, 1993; Sigal & Tatum, 1988). Such cross-reactivity could contribute to chronic B. burgdorferi-triggered, immune-mediated symptoms. Other researchers (Alaedini & Latov, 2005) found cross-reactive human neural epitopes that share amino acid sequences with B. burgdorferi OspA protein; antibodies against OspA peptides reacted in vitro with neurons in human brain, spinal cord and dorsal root ganglia. Elevated levels of antineuronal antibodies have been reported in two samples of patients with PTLDS (Chandra et al., 2010). Guillain-Barre Syndrome – a progressive life-threatening antibody-mediated neurologic disorder characterized by progressive ascending weakness– can be triggered by B. burgdorferi-infection and has been shown to response to ceftriaxone and intravenous gammaglulin therapy (IVIg) (Patel, Shah, & Subedi, 2017). Cases and small series have reported benefit to IV Ig treatment for patients with neuropathic symptoms after Lyme disease, based on the poor response to antibiotics and a known benefit of IVIg therapy for autoimmune neuropathies (Katz & Berkley, 2009; Rupprecht, Elstner, Weil, & Pfister, 2008). Summary Lyme disease in humans produces a multi-systemic, multi-staged illness and while disease manifestations have been well characterized, host-pathogen interactions and the resultant pathophysiology are poorly understood. The number and severity disease manifestations vary greatly from patient to patient suggesting that host determinants play a role. Tissue tropism (bacterial preference for certain tissues) is notable but factors driving this selectiveness is unknown. Many of the most challenging symptoms and signs that patients contend with are neurological in nature yet how B. burgdorferi interacts with the nervous system is uncertain. Possibilities include 1) the release of lipoproteins into the circulation that cross the blood brain barrier and act via inflammation or direct effects on neural tissue, 2) pro-inflammatory cytokines, 3) formation of autoantibodies and/or cross-reactive antibodies. The pathophysiology underlying ongoing symptoms and signs in patients previously treated for Lyme disease is not understood. Potential mechanisms include immune dysfunction, tissue injury, untreated co-infections and persistent B. burgdorferi infection; these mechanisms are not mutually exclusive. Given the uncertainty regarding human pathophysiology in Lyme disease, continued research is required as an increased understanding of the infection should translate to improved patient care. Gaps in Knowledge As is known from the literature and the clinical experience of doctors who diagnose and treat patients with Lyme disease, patients can have many different disease manifestations. Diagnosis and treatment is made more difficult because the pathogen itself can only rarely be detected in the blood, early diagnosis does not always occur, and pathogen eradication cannot be proven following treatment. There are many gaps in the scientific understanding of the pathophysiology of Lyme disease. In considering these gaps, the subcommittee has categorized them as either related to disease manifestations in humans and gaps regarding what is known about the pathogen(s). Pathogen(s): Do different B. burgdorferi strains and/or variants affect the rate of infection, course of disease and/or disease manifestations pre and post-treatment? In animal studies, different strains can cause different signs of disease (Cerar, Ogrinc, Strle, & Ruzic-Sabljic, 2010). It is also known that of the twenty different species in the B. burgdorferi sensu lato complex, eight are pathogenic in humans and several of these species have been shown to cause disease in the U.S. (Clark, Leydet, & Threlkeld, 2014). Do some of these strains cause worse neurological manifestations or cardiac involvement and might it be worthwhile to identify infections to the strain level? In other infectious diseases such as HIV/AIDS, identifying the specific viral pathogen guides treatment, having the same degree of detail in Lyme disease could provide clinicians with important information regarding treatment options. Is persistent infection with B. burgdorferi responsible for persistent disease? As detailed previously in this report, there is evidence in multiple species (non-human primates, mice, dogs, rabbits) that B. burgdorferi can survive antibiotic exposure. In a study of non-human primates, B. burgdorferi survived 12 weeks of antibiotic exposure (Embers et al., 2012). There are also human case reports of persistent infection following short and longer antibiotic regimens (see Appendix 3). Patient treatment outcomes after certain antibiotic regimens support the hypothesis that persistent disease is due to persistent infection. However, it is not known if this persistent infection is the cause of symptoms in humans. Alternatively, does the immune system's reaction to these remaining spirochetes or antigenic debris become dysregulated and generate the symptoms that patients feel? After what duration of infection does this happen? What assays and/or approaches might be developed that could reliably detect the presence and absence of B. burgdorferi? Diagnostic tests that are based on the antibody response to B. burgdorferi are often unreliable for identifying active infections. For example, serology is insufficiently sensitive in the acute stage if testing occurs before antibodies have formed. It is also is uninformative following antibiotic therapy as positive results are not necessarily indicative of infection and negative results do not necessarily indicate that the infection has been cleared. Methods that detect the pathogen itself, irrespective of strain, species or duration of infection, would benefit clinicians and patients. Attention should also be paid to developing assays in alternative body fluids like cerebrospinal fluid, as detecting and treating neurological infections early in the disease may be important in reducing the likelihood that a patient would continue to experience signs and symptoms of disease following treatment. Does delayed diagnosis of B. burgdorferi infections affect the rate and/or quality of persistent symptoms? What are the mechanisms by which diagnostic and treatment delays leads to persistent symptoms? What is the rate of co-infections in patients infected with B. burgdorferi and do co-infections affect treatment outcome? Studies assessing tick infection rates of different pathogens demonstrated that ticks can be infected with one or more pathogens, including co-infections with different strains of B. burgdorferi. What can we learn from other sources of data, such as patient registries or EMR databases? Might there be a correlation between multi-pathogen infection and disease presentations and patient outcomes? Human Hosts What predisposing factors in the host contribute to a greater risk for persistent symptoms? Are there other genetic or epigenetic or clinical factors in patients that contribute to persistent symptoms? Does an aberrantly activated central neural response to B. burgdorferi impact symptom persistence? What are the pathophysiologic mechanisms underlying this continued neural dysregulation? Several brain imaging studies have shown that some regions of the brain have decreased metabolism and blood flow in patients with chronic symptoms. In some, but not all cases, these aberrations were reversed with antibiotic therapy. The hypersensitivity to light and sound and the increased pain experience by many patients with ongoing symptoms following antibiotic therapy may be due to central sensitization - mediated by circuits in the brain that are aberrantly activated. Are there aberrantly activated neural circuits in this group of patients? Once identified, could treatment focused on these neural networks resolve chronic symptoms? What are the effects of B. burgdorferi infection on the immune system and do those effects contribute to clinical disease? Multiple studies in animals show that B. burgdorferi alters both the innate and acquired immune response and can persist despite a competent immune response. It is known that antibiotics do not eradicate infection on their own but hold the infection at bay until the immune system can mount a more robust response. If B. burgdorferi alters the immune response in humans, in longstanding infections, could the immune response be altered to such a degree that despite the use of antibiotics, the immune response is unable to eradicate the bacteria? Does infection with B. burgdorferi trigger autoimmune disease? Recent studies indicate that some patients with Lyme arthritis (Arvikar, Crowley, Sulka, & Steere, 2017) go on to develop autoimmune arthritis, psoriatic arthritis, spondyloarthritis, or rheumatoid arthritis after Lyme disease. In addition, the development of thyroid disease appears to be higher in Lyme patients than the general public (Gelburd, 2017). Gaps in our understanding of how Borrelia burgdorferi impacts the brain Large epidemiologic studies are needed to clarify the frequency of cognitive impairment and other neurologic and neuropsychiatric sequalae after B. burgdorferi infection and to determine whether the frequency of these sequalae in Lyme disease patients is greater than among those individuals who do not have a history of Lyme disease. Comprehensive studies of children with Lyme disease – both cross-sectional and prospective – are needed to better understand potential pediatric-specific manifestations, the impact of the infection on developmental, and long-term outcomes. There is a need to take advantage of the advanced neuroimaging technologies to better understand persistent symptoms such as “brain fog”, “sensory hyperarousal”, “chronic pain”, or “depression”. Neuroimaging has advanced considerably in the last decade and has been helpful in clarifying the pathophysiology of other diseases. As the pathophysiology is clarified and the specific areas or circuits of abnormality mapped, targeted treatment interventions can then be applied. A few indications and examples are included in the following: To identify dysfunctional brain circuits. Functional Magnetic Resonance Imaging studies can be done to clarify whether patients have abnormally functioning neural circuits; for example, a fMRI pain study could help clarify the brain areas involved in the heightened sensory arousal of patients and reveal whether these areas are hyper-activated, consistent with the model of central sensitization. To assess inflammation or tissue proteins or neurotransmitter transporters or receptors. Molecular Imaging using Positron Emission Tomography (PET) scanning with radioactive ligands bind to receptor targets of choice. For example, a tracer may bind to microglia – indicating microglial activation (for example, inflammation). To clarify biochemical abnormalities. Magnetic resonance spectroscopy can reveal chemical/metabolic components in both the CSF and the brain tissue that reflect neural integrity. Potential Actions: Action 1: Perform clinical trials that use more inclusive criteria, are more representative geographically, and include co-infected patients, to determine appropriate therapeutic regimens to resolve the diverse manifestations of Lyme disease Clinical trials to assess diagnosis and treatment in Lyme disease are difficult to perform. Patients present with differing duration of illness, symptomology and rash presentations, as well as different medical conditions and potentially different co-infections (whether known or unknown). The trials are usually performed only in highly endemic areas (Northeast) despite the significant burden of disease in the Midwest, South and West. Most Lyme trials were completed decades ago with little long-term follow-up, before the diversity of the illness and the potential for delayed pathophysiology were recognized. The understanding of the regionality of strains and species continues to evolve. Other Borrelia burgdorferi sensu lato species are now known to infect humans (B. mayonii, B. bissettiae) in North America. This is may have significant implications as animal and human studies confirm that different strain/species can produce different manifestations which may necessitate either shortening or lengthening treatment regimens. In addition, entry into a Lyme disease clinical trial most often requires a patient to have an EM or an objective sign of disseminated disease that meets the CDC surveillance criteria. Positive two-tiered serologic testing may also be required, potentially disqualifying 50% of potential participants. Excluding seronegative Lyme patients from studies may produce an incomplete picture of the best methods to diagnose and treat all patients. For example, 17% of subjects in a Lyme encephalopathy trial were seronegative but were included on the basis of other testing (Logigian et al., 1999). Ensuring study of the broader Lyme disease population, not just those who meet the CDC surveillance case definition, will enable a more accurate assessment of diagnostic sensitivity and patient treatment regimens. As Dr. Burgdorfer demonstrated, ticks are often co-infected with other bacteria, viruses, and parasites. Ticks can also be simultaneously infected with multiple B. burgdorferi strains or Borrelia species (Crowder et al., 2010). Multiply-infected patients may have different disease presentations, symptomology, and treatment responses; some may require antimicrobials that are distinctly different from those used in Lyme disease. To date, no prospective studies have been conducted in patients who are simultaneously infected with more than one tick-borne pathogen. It is imperative to support research which studies the effect of simultaneous infection with multiple tick borne pathogens in humans. Action 2: Clinician education: Provide more comprehensive clinician education that highlights diverse symptomology, expanding geography of infecting ticks, and limitations of current testing regimen Disease manifestations in Borrelia infection are numerous and span most major body systems. Because of diverse and migrating clinical symptomology, patients with Lyme disease can present to many different primary care and specialist clinicians in the outpatient or inpatient setting – for example, internal/family medicine, pediatrics, emergency medicine, cardiology, rheumatology, neurology, and psychiatry. Patients may not recall having a tick bite or EM rash, and patients may not present until months or years after the onset of the infection. Insensitive diagnostics and the inappropriate use of test results add to the confusion. While Lyme disease may be more often recognized in highly endemic areas due to frequent patient presentation and focal tick activity seasons, current studies show a 44% increase in counties with Ixodes species ticks in the 43 states known to harbor them (Eisen, Eisen, & Beard, 2016). Many of those counties are outside of the area considered “endemic” for Lyme disease. In addition, some people from non-endemic states acquire Lyme disease during travel to endemic areas but don’t become symptomatic until they return home and, thus, present to a clinician who may not have extensive experience diagnosing or treating the infection. While doctors outside the top fourteen endemic states may not encounter many cases of Lyme disease, it is important that they be able to recognize the diverse signs and symptoms of the infection so as to not miss the diagnosis. Clinician education is crucial to the early diagnosis of Lyme disease as well as to the prevention of disease progression and poor patient outcomes. A thorough assessment of medical school education on tick-borne diseases for all medical school students is warranted. As noted in several places in our report, very few human pathology studies of patients with Lyme disease have been completed. Much of what is known about the pathology and pathophysiology of Lyme disease has been learned from animal models and may not be fully representative of human disease. Results for Priority 3–The Treatment of Lyme Disease Question 1: What is/are the best treatment regimens for acute Lyme disease, and for patients with ongoing symptoms who have or have not been previously treated? Given that the estimate of annually occurring new cases of Lyme disease in the US exceeds 300,000 and the costs associated with both successful and unsuccessful antibiotic interventions as well as palliative therapies, the imperative to identify curative/restorative therapies for all stages of Lyme disease is quite clear (Adrion et al., 2015; Hinckley et al., 2014; Johnson, Wilcox, Mankoff, & Stricker, 2014; Klempner et al., 2001; Zhang et al., 2006). Identifying best regimens is a values-driven analysis. In healthcare, the primary goal is to improve patient lives by optimizing the outcomes that are important to them. To be effective, therapies must provide patients with treatment benefits that positively effect how they feel, function, or survive. A number of government and non-government organizations including, the National Academy of Medicine (NAM, formerly the Institute of Medicine (IOM)), the Agency for Healthcare Research and Quality (AHRQ), the Food and Drug Administration (FDA), and the Patient-Centered Outcomes Research Institute (PCORI), support patient-centered research and its implementation in healthcare, which emphasizes individualized care, consideration of patient values and preferences among treatment alternatives, and shared decision making in encounters between physicians and patients (Clinical Practice Guidelines We Can Trust, Institute of Medicine. 2011; Crossing the Quality Chasm: A New Health System for the 21st Century, Institute of Medicine. 2001; PCORI, 2017). Patient-centered research emphasizes involving patients as pivotal-stakeholders over the entire research spectrum: from identification of research question to dissemination of results. Patient-centered care, through shared decision making and patient involvement in patient-centered outcomes research, is considered an important feature of value-based care (Jayadevappa, 2017). Most traditional medical research has been investigator-driven using objective disease-centered outcomes selected by investigators as primary end points. As clinical research has evolved, research design and implementation include active engagement of both investigators and patients, to employ outcomes measures that are important to both patients and investigators. For example, the resolution of an erythema migrans rash is an objective disease specific measure often used as a primary endpoint in early Lyme disease clinical trials. However, the outcome that patients care most about is resolution of their symptoms and the ability to resume the life they led before becoming ill. Similarly, a primary outcome for Lyme meningitis is the elimination of signs of active infection in the spinal fluid. While this is a valuable end point, the patient’s concern will be to measure resolution of headaches and of fatigue which has a direct impact on patient’s quality of life and ability to function; these patient-centered outcomes are often relegated to status as a secondary outcome measure and not used to determine the efficacy of a treatment (see Appendices 5 and 6). A treatment would be labeled as effective, but the patient might have fatigue lasting months or years. With regard to patient centered outcomes for Lyme disease, an Outcomes Important to Lyme Patients Survey of over 6000 Lyme disease patients conducted by LymeDisease.org in March 2015 (MyLymeData: Highlights from Pateint Registry Data 2018, publication pending)(NCHS, 2017) identified several outcomes of importance, including: symptom reduction, quality of life improvement, reducing functional impairments, and avoiding disease progression (see Appendix 6). Most patient reported outcome measures used today, were developed by expert panels. Although many diseases establish a disease-specific common core set of outcomes measures for clinical trials, this has not been developed for Lyme disease studies, which have typically relied on general standardized patient reported outcomes measures that were developed for use across different diseases. A Core Outcome Set is an agreed minimum set of outcomes or outcome measures that is a recommendation of 'what' should be measured and reported in all trials in a specific area. A Core Outcome Set a) can be developed with a patient community, b) allows the same outcomes to be measured across different trials, and c) eliminates non-relevant items and domains included in generic patient reported outcomes measurement tools. In the clinical setting, patient centered care focuses on shared medical decision making that takes into account the individual circumstances and values of the patient. When the evidence base is uncertain and where different combinations of treatment options, uncertain outcomes and implicit trade-offs exist, patient involvement is critical to make the “right choice” (Hirsch, 2018; Sox & Greenfield, 2010). Under shared decision making, clinicians are viewed as the experts in the evidence and patients are the experts in what matters most to them (Spatz, Krumholz, & Moulton, 2017). In the outcomes survey, patients valued identification of treatment options and the risks and benefits associated with them as well as shared medical decision making. Evidence Pertaining to the Treatment of Lyme disease Ideally, the provision of evidence-based, patient centered care is built on a solid research foundation of well-designed and conducted clinical trials employing patient-centered outcomes. With regard to the treatment of Lyme disease in the United States, the evidence base does not reflect the ideal. The available US-conducted clinical trial evidence is not robust. There were few randomized trials and of these, many had design and/or execution flaws; all had relatively small sample sizes (Cadavid, Auwaerter, Rumbaugh, & Gelderblom, 2016; Cameron, Johnson, & Maloney, 2014; Hayes & Mead, 2004; NICE, 2018; Wormser et al., 2006). The treatment of many common presentations of Lyme disease has not been investigated in randomized clinical trials or observational studies. In these situations, the treatment protocols that have emerged are based on case reports and expert opinion. Although there are a number of evidence assessment processes, more than 70 organizations have adopted the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology for guideline development. These organizations include Centers for Disease Control and Prevention (CDC) Advisory Committee on Immunization Practices (ACIP), Agency for Health Care Research and Quality (AHRQ), American College of Physicians (ACP), American Academy of Family Physicians (AAFP). GRADE emphasizes separating evidence assessments from recommendations as well as the values and preferences of patients considering those alternatives. In addition, it encourages shared decision making between physicians and patients (Guyatt et al., 2013). GRADE initially ranks randomized controlled trials the highest but downgrades them based on a) limitations in design (e.g. large loss to follow-up), b) inconsistency (e.g. conflicting results across trials), c) indirectness (e.g. lack of generalizability to clinical population), d) imprecision (e.g. small sample sizes or where the minimum clinically important treatment effect is unknown and e), reporting bias (e.g. when certain results are more likely to be published). Observational studies are initially ranked low but may be upgraded based on treatment effect size. For evidence that is regarded to be of low or poor quality, further research is very likely to have an important impact on the estimate of treatment effect. Several reviews of the evidence have been completed. Four were GRADE-based ‒ Hayes and Mead (Hayes & Mead, 2004), International Lyme and Associated Diseases Society (ILADS) (Cameron et al., 2014), Cochrane (Cadavid et al., 2016), and England’s National Institute for Health and Care Excellence (NICE) (NICE, 2018). A fifth review was conducted by the Infectious Diseases Society of America (IDSA) (prior to the organization adopting GRADE); it included retrospective studies that the others did not (Wormser et al., 2006). The Hayes and Mead and Cochrane assessments were limited to neuroborreliosis; the ILADS assessments focused on the treatment of erythema migrans and persisting, post-treatment manifestations. The NICE assessments and the IDSA guidelines review examined the most common presentations of Lyme disease (Hayes & Mead, 2004; ILADS, 2016; "ISDA on Lyme," 2005; NICE, 2018). The NICE and Cochrane GRADE assessments as well as the IDSA review included trial evidence conducted outside the US. However, given the differences in the pathogenicity and virulence between genospecies in the B. burgdorferi sensu lato group as well as differences between European and US strains of B. burgdorferi, this report primarily considers US-conducted trials. In general, the four GRADE-based evidence assessments focused exclusively on randomized trials and found the evidence quality to be low or very low (with the exception of the fatigue finding in the Krupp trial, which was moderate). The IDSA guidelines review of the evidence quality typically awarded higher ratings than the GRADE-based assessments, demonstrating that inconsistencies between ranking systems exist. The GRADE-based assessments reduced the quality rating of randomized controlled trials when there was evidence of bias in trial design or execution, imprecision or indirectedness (population studied/trial circumstances differed from situations of intended use) whereas the IDSA review did not. The discussion of treatment in this report leans heavily on the aforementioned assessments and review, supplementing them when appropriate. Research Gaps/Opportunities (Patient Centered): 1. Can a patient-centered core outcome set be developed for clinical trials of management of Lyme disease? Core outcome sets are an agreed minimum set of outcome domains to be measured and reported in all trials of a particular treatment or condition. They help ensure consistency in outcomes measures across trials and that the outcome measures pertain to matters that patients regard as important. 2. How do patients regard the value of specific outcomes or treatment tradeoffs? Treatment values and preferences among patients vary considerably when considering treatment alternatives. Understanding the range of preferences helps to shape shared medical decision-making. 3. What magnitude of treatment effect is clinically meaningful to patients? Research trials in Lyme disease have been criticized for using unduly large treatment effects to measure effectiveness of treatment (Delong, Blossom, Maloney, & Phillips, 2012). Patient-centered research should determine the minimal treatment response rate that is clinically relevant and meaningful to patients. 4. What is the best way of using shared medical decision-making tools to provide evidence about treatment options (including the option of not treating) and the risks and benefits associated with those options to help clinicians and patients make informed decisions about care? Shared medical decision making should be used when there are evidence gaps, when no single treatment approach is clearly superior, and when patients may value different treatment trade-offs in the context of their particular circumstances (NLC, 2013). 5. How can we achieve greater patient engagement throughout the research process? In the past Lyme disease treatment studies have had limited or no patient engagement. The new research paradigm recognizes that patient engagement in research is essential to ensure that the research question is meaningful to patients, the trial design is patient centric, recruitment efforts are successful and that the translation of research into practice is faster. Treatment Duration in Early disease Seven comparative trials investigated the treatment of early Lyme disease with either amoxicillin, azithromycin, cefuroxime or doxycycline (Dattwyler, Volkman, Conaty, Platkin, & Luft, 1990; Eppes & Childs, 2002; Luger et al., 1995; Massarotti et al., 1992; Nadelman et al., 1992; Wormser et al., 2003). Noncompletion rates exceeded 20% in two studies (Luger et al., 1995; Wormser et al., 2003), and in individual arms of two others (Eppes & Childs, 2002; Nadelman et al., 1992). Three observational studies are also considered here (Aucott, Rebman, et al., 2013; Gerber, Shapiro, Burke, Parcells, & Bell, 1996; Smith et al., 2002); retrospective studies and a comparative trial of erythromycin, penicillin and tetracycline were not. The latter is a much older study that included agents that are no longer widely used (Steere et al., 1983). Retrospective studies have an increased risk of biasing findings and, thus, were not considered. Trial designs were highly variable in terms of agents compared, duration of therapy and definitions of success and failure (Aucott, Rebman, et al., 2013; Dattwyler et al., 1990; Eppes & Childs, 2002; Gerber et al., 1996; Luft et al., 1996; Luger et al., 1995; Massarotti et al., 1992; Nadelman et al., 1992; Smith et al., 2002; Wormser et al., 2003). None of the trials simultaneously compared all four agents. Resolution of the EM rash was the primary endpoint in all studies. Resolution of symptoms, a patient-centered outcome, was a secondary endpoint. The median duration of treatment in the individual studies ranged from 10 - 30 days. In many studies, subjects were retreated when the initial therapy failed (Aucott, Rebman, et al., 2013; Eppes & Childs, 2002; Gerber et al., 1996; Luft et al., 1996; Luger et al., 1995; Massarotti et al., 1992; Nadelman et al., 1992). The individual trials were small. Subset analysis was not routinely done but some investigators associated certain presenting symptoms/signs such as multiple EM, paresthesias, dysesthesias, irritability and/or increased severity of initial symptoms with an increased risk of treatment failure (Luft et al., 1996; Luger et al., 1995; Massarotti et al., 1992; Nadelman et al., 1992). In an attempt to provide meaningful information regarding the relative effectiveness of the different first-line agents, the trials were reanalyzed using symptom resolution as the primary outcome. Trials or arms that had noncompletion rates ≥ 20% were excluded because that degree of missing data raises uncertainty about the outcome findings (Fitzmaurice, Laird, & Ware, 2004). Patients who remain symptomatic or who failed to complete the trial were categorized as failures. While this analysis is imperfect, the results are worth considering. Based on the data for the studies final observation, the success rates for resolution of EM and associated symptoms are: amoxicillin 87-89%, azithromycin 75%, cefuroxime axetil 62%, doxycycline 87%. See Table 8 for the results of this analysis. Table 8. Patient-centered, Adjusted Outcomes in the Treatment of Erythema Migrans Trial; Duration of Therapy Amoxicillin Azithromycin Cefuroxime Doxycycline - S F S F S F S F Dattwyler; 21d (Dattwyler et al., 1990) - - - - - - 35/37 (95%) 2/37 (5%) Massarotti; Az-5d; dox -10 (Massarotti et al., 1992) - - 13/16 (81%) 3/16 (19%) - - 14/22 (64%) 8/22 (36%) Nadelman 20d (Nadelman et al., 1992) - - - - 34/63 (54%) 29/63 (46%) - - Luft; Am-20d; Az-7 (Luft et al., 1996) 87 – 90/122 (71-74%) 32 – 35/122 (26-29%) 92/124 (74 %) 32/124 (26%) - - - - Eppes; 20d (Eppes & Childs, 2002) 12/13 92% 1/13 8% - - 14/15 (93%) 1/15 (7%) - - Gerber; 21d (Gerber et al., 1996) 135-136/137 (98-99%) 1-2/137 (0.7-1.5%) - - - - 50-51/51 (98-100%) 0-1/51 (0-2%) Smith; Am-30d, dox – 22d (Smith et al., 2002) 29-30/30 (97-100%) 0-1/30 (0-3%) - - - - 79-80/81 (98-99%) 1-2/81 (1-2%) Aucott; 21d (Aucott, Rebman, et al., 2013) - - - - - - 42/63 (67%) 21/63 (33%) Total 263-268/302 (87-89%) 33-38/302 (11 -13%) 105/140 (75%) 35/140 (25%) 48/78 (62%) 30/78 (38%) 220-222/254 (87%) 32-34/254 (13%) S = success, F = failure, Am = amoxicillin, Az = azithromycin, dox = doxycycline Percentages refer to percent of success or failure for each antibiotic regimen Overall, the majority of patients treated with ≥ 20 days of antibiotic had resolution of their EM rash and symptoms. The same cannot be said for patients treated with 10 days of doxycycline. In the doxycycline arm of the Massarotti et al trial, seven of the 22 subjects were immediately retreated with 10 additional days of oral antibiotics and an eighth patient was subsequently retreated with ceftriaxone (Massarotti et al., 1992). Thus, this regimen failed in 36%. Educational Gaps/Opportunities: Clinicians may be unaware that blood tests at this stage should not delay the initiation of antibiotics. Clinicians may be unaware of the details regarding the efficacy of the different antibiotic regimens or that unless contraindicated, doxycycline is preferred because it is effective against other blacklegged tickborne pathogens such as Anaplasma phagocytophilum, Borrelia miyamotoi and Borrelia mayonii. Clinicians may be unaware that some researchers retreated subjects who remained ill, relapsed or experienced disease progression. Research Gaps/Opportunities: Develop a core set of outcome measures that are patient-centered. How can we reduce failure rate among those treated for early Lyme disease? Are certain subgroups of EM patients at higher risk for treatment failure and if so, how might they be identified? Is there a difference in long-term efficacy between 10,14 and 21-day regimens of doxycycline, cefuroxime or amoxicillin? Would longer term initial therapy reduce the percentage of patients who develop later stage disease despite early antibiotic therapy? Treatment of Lyme Carditis No trials or observational studies have been conducted on the treatment of Lyme carditis in general or with regard to specific manifestations of cardiac involvement. Long-term follow-up studies have not been conducted. Borrelia burgdorferi can infect any or all layers of the heart; the most commonly reported manifestation of cardiac involvement is atrio-ventricular (AV) block (Forrester & Mead, 2014). Such reporting may not reflect the actual relative incidence of cardiac manifestations as the CDC surveillance case definition of Lyme carditis only recognizes patients who have 2° or 3° AV block and positive two-tier serology. Regardless, as highlighted by the CDC paper on sudden deaths due to Lyme carditis (CDC, 2013), AV block is a potentially lethal consequence of infection with B. burgdorferi and management of this condition requires careful consideration. Epidemiologic data suggests that males are at higher risk for Lyme carditis in general and AV block in particular (Forrester & Mead, 2014). Although there have been no studies regarding the treatment of Lyme carditis, the IDSA guidelines and the NICE GRADE assessments have made recommendations for the treatment of Lyme carditis which this subcommittee supplements with its own recent literature review (NICE, 2018; Wormser et al., 2006). A baseline ECG should be obtained for all patients with cardiorespiratory symptoms and considered for children 10 years of age and older who have either been diagnosed with Lyme meningitis or who have disseminated disease and arthralgia as these groups are at higher risk of Lyme carditis (Costello, Alexander, Greco, Perez-Atayde, & Laussen, 2009; Forrester & Mead, 2014; Welsh et al., 2012). Patients who are hemodynamically unstable as well as those with syncope, dyspnea or chest pain, patients with 1⁰ block with a PR interval ≥ 30 milliseconds, 2⁰ or 3⁰ AV block, atrial fibrillation with prolonged and/or frequent sinus pauses, severe left ventricular dysfunction or severe valvular dysfunction should also be hospitalized and closely monitored as the progression in AV block can occur rapidly (NICE, 2018; Wormser et al., 2006). Inpatient therapy largely relies on IV ceftriaxone, 2 g once daily, which may be changed to oral doxycycline at the time of discharge. Treatment durations are similar, the IDSA guidelines recommends 14-21 days and the NICE GRADE assessment recommends 21 days. Although there have been no studies regarding the long-term outcomes of outpatient treatment of Lyme carditis, the IDSA guidelines recommends 14-21 days and the NICE GRADE assessment recommends 21 days (NICE, 2018; Wormser et al., 2006). In the absence of contraindications, outpatient therapy generally relies on doxycycline, 100 mg twice daily because it covers other blacklegged tick-transmitted diseases that may have be present. Amoxicillin, 500 mg three times daily and cefuroxime 500 mg twice daily may be used as alternatives. Pediatric dosages are the following: ceftriaxone, 50–75 mg/kg IV once daily to a daily maximum of 2 g; amoxicillin, 50 mg/kg per day in 3 divided doses to a maximum of 500 mg per dose; cefuroxime axetil, 30 mg/kg per day in 2 divided doses to a maximum of 500 mg per dose. Antibiotics that can affect the QT interval, such as macrolide antibiotics, should be avoided (NICE, 2018). Educational Gaps/Opportunities Clinician may be uninformed regarding the presentation and identification of Lyme carditis and may not appreciate that this is a potentially lethal condition. Research Gaps/Opportunities: What are the long-term outcome for patients who were treated for Lyme carditis? Could Lyme disease result in a dilated cardiomyopathy as suggested by Kuchynka et al (Kuchynka et al., 2015) and Klein et al (Klein et al., 1991). If so, this would have broader implications with regard to diagnostic testing and treatment in patients with dilated cardiomyopathy of unknown etiology. Lyme Arthritis Several randomized trials and observational studies have been conducted on the treatment of Lyme arthritis. The NICE GRADE assessment considered three (NICE, 2018); the IDSA review considered two of these and several others (Wormser et al., 2006). The management recommendations from both groups are similar. Adults and children with uncomplicated Lyme arthritis (no neurologic involvement) should receive 28 days of an oral antibiotic. Doxycycline, 100 mg twice daily is the preferred regimen. If there are contra-indications to its use or drug intolerance, amoxicillin, 500 mg three times daily or cefuroxime axetil 500 mg twice daily may be substituted. Pediatric dosages are as follows: amoxicillin, 50 mg/kg per day in 3 divided doses to a maximum of 500 mg per dose; cefuroxime axetil, 30 mg/kg per day in 2 divided doses to a maximum of 500 mg per dose. If joint pain and swelling persist, a second course of oral antibiotics may be prescribed. Alternatively, a 2-4-week course of IV ceftriaxone may be used. Patients whose arthritis is accompanied by neurologic manifestations should immediately receive IV ceftriaxone to avoid the development of overt neuroborreliosis (Wormser et al., 2006). Educational Gaps/opportunities: Physicians may not know that patients with Lyme arthritis should be carefully evaluated for neurologic signs and symptoms of Lyme disease and that ceftriaxone is the agent of choice if neurologic manifestations are identified. Research Gaps/opportunities A need to identify markers for the management of antibiotic refractory Lyme arthritis. Treatment of Neuroborreliosis Three GRADE assessments (NICE, Cochrane and Hayes and Mead) and the IDSA guidelines evaluated the evidence for the treatment of neuroborreliosis. The GRADE assessments found the evidence to be low or very low while the IDSA review rated evidence quality as II on a I – III scale. The 2016 Cochrane Grade assessment search yielded no US-conducted antibiotic trials for neuroborreliosis. Given the recognized differences between US and European genospecies and strains of B. burgdorferi as well as differences in European and US presentations, this report does not consider the Cochrane report and only considers the portion of the NICE assessment dealing with US trials. Thus, the IDSA guidelines and a literature review by this panel inform the management discussion which follows. For meningitis, the IDSA guidelines recommend IV regimens such as ceftriaxone, 2 g daily (cefotaxime 2 g every 8 hours is an alternative) for 10-28days (Wormser et al., 2006). Facial and other cranial nerve palsies that lack signs of meningitis may be treated with oral antibiotics such as doxycycline or amoxicillin for 14-21 days (Wormser et al., 2006). (The evidence is limited to patients with facial nerve palsy whether it should be extrapolated to other types of cranial neuropathy is unknown). For the treatment of later neurologic manifestations, central or peripheral nervous system disease, the IDSA guidelines recommend IV ceftriaxone (cefotaxime is an alternative) for 14-28 days and does not recommend retreatment unless there is objective evidence of relapse (Wormser et al., 2006). This recommendation is at odds with the recommendation that allows for retreatment of Lyme arthritis and the available evidence. Two observational studies by Logigian et al documented many of the impacts of neurologic disease (Logigian et al., 1990, 1999). In the first study, 27 patients received 2 g of IV ceftriaxone for 14 days. At the final 6-months post-treatment follow-up visit, 17 (63%) were improved, 6 (22%) had improved and then relapsed and 4 (15%) were unchanged. In the second study 18 patients with Lyme encephalopathy received 2 g of IV ceftriaxone for 30 days. At the final assessment (an average of 22 months post-treatment), 7 (39%) reported they were normal, 9 (50%) were greatly improved, and 2 (11%) were somewhat improved. (“Greatly” and “somewhat” were undefined) One patient who relapsed was subsequently retreated and was improved at the final assessment. In general, these two studies demonstrated that patients who received 14 days of ceftriaxone fared worse than those who received 30 days. Relapse was significantly higher in the 14-day group and there was no response to therapy in 4 (9%). Given that patients value a return to health and minimizing the impact of disease, the overall findings from the Logigian trials discussed above, including the positive response to retreatment in a subject who relapsed, and the IDSA recommendation that arthritis may be retreated (Wormser et al., 2006), the concept of not offering retreatment to patients with neurologic disease appears to be out of step. Educational Gaps/Opportunities Clinicians may be unaware that the onset of neurologic manifestations may be delayed for years (Logigian et al., 1990). Research Gaps/Opportunities In the treatment of acute neurologic Lyme disease (such as meningitis, radiculopathy, facial nerve palsy with likely central nervous system involvement), is oral doxycycline comparable to IV ceftriaxone? Is 14 days of treatment as efficacious as 28 days? Treatment of Late Lyme Disease with Symptoms and Signs not included in the CDC Surveillance Case Criteria Some patients with late Lyme disease present with symptoms and signs that do not fit within the surveillance case definition. Examples of such patients would be people with Lyme disease who do not demonstrate the following clinical signs: carditis (defined as the acute onset of 2⁰ or 3⁰ AV defects that resolve in days to weeks and are sometimes associated with myocarditis); recurrent, brief attacks of arthritis; lymphocytic meningitis, cranial neuritis, radiculoneuropathy, rarely, encephalomyelitis (CDC, 2017). Patients who do not present with the CDC surveillance criteria signs may experience a delay in their diagnosis (See Appendix 7); this delay allows the B. burgdorferi spirochete to become host-acclimated prior to the start of antibiotic therapy. Common symptoms include fatigue, headache, pain, myalgia, arthralgia and cognitive impairment, sleep disorders, psychiatric symptoms and parasthesias (See Figure 3). There have been no controlled studies on this patient group; published reports of US-conducted observational studies are discussed below (Donta, 2002, 2003). The underlying pathophysiology for these symptoms is unknown. While the biologic basis for persistence of B. burgdorferi remains to be determined, it is known that the organisms can elude and/or alter the normal host immune responses. This allows the bacteria to persist and perhaps to find a locus to avoid further host immune responses. It has been assumed that there are no intracellular reservoirs for B. burgdorferi but an alternative hypothesis has been proposed (Donta, 2003). This hypothesis suggests that B. burgdorferi take up residence in intracellular endosomes (altering them in some manner, which could include changes in endosomal pH and normal phagolysosome trafficking mechanisms) as a means of intracellular survival. In vitro and clinical observations that certain antibiotics that are capable of intracellular penetration are more effective in eradicating B. burgdorferi than antibiotics that do not support the possibility of an intracellular reservoir for these borrelia (Donta, 2003; Levin et al., 1993). For example, clarithromycin, which is highly active against B. burgdorferi in vitro, and achieves high intracellular levels, is ineffective or poorly active clinically in treating patients with persistent Lyme disease. But when hydroxychloroquine, an intracellular alkalinizing agent, which when given alone to patients with ongoing signs and symptoms of antibiotic treated Lyme disease is also ineffective, is combined with clarithromycin, patients respond well to this combination of medications(Donta, 2003). However, hydroxychloroquine as a single agent is recommended for antibiotic-refractory Lyme arthritis (Steere et al., 2017; Steere et al., 2016). The pharmacologic explanation lies in the fact that during the process of intracellular trafficking of internalized bacteria, endosomes are acidic and clarithromycin is not active in an acidic milieu (Donta, 2002, 2003). Hydroxychloroquine alkalinizes such endosomes, conferring greater antibiotic efficacy for clarithromycin (Donta, 2003). Further support for this explanation is provided by the observation that that the activity of tetracycline, which is active in an acidic milieu, is not affected by the addition of hydroxychloroquine to the treatment regimen (Donta, 2012). (Potential reasons that the PLEASE trial did not demonstrate treatment efficacy for the clarithromycin/hydroxychloroquine combination in treating patients with Lyme disease are discussed subsequently in this paper.) Educational Gaps/Opportunities Some clinicians may not be aware that patients may present with only nonspecific symptoms and signs of Lyme disease. Research Gaps/Opportunities What is the optimal dose, optimal route of administration, type of antibiotic or combination of antibiotics for treating late stage Lyme disease that has not been previously treated? To what extent does B. burgdorferi alter the normal human immune response? Whether Bb impairs the ability of the human host to completely clear the infection following the initiation of antibiotic treatment is an important outstanding question. Treatment of Patients with Ongoing Symptoms or Signs of Lyme Disease Despite Prior Antibiotic Therapy Four NIH sponsored antibiotic retreatment trials of patients with posttreatment Lyme disease were conducted in the United States (Fallon et al., 2008; Klempner et al., 2001; Krupp et al., 2003). In addition to the GRADE assessments by ILADS and NICE and the IDSA review, other reviews on this topic were also considered (Delong et al., 2012; Fallon, Petkova, Keilp, & Britton, 2012; Klempner et al., 2013). In general, the two GRADE-based assessments found the evidence quality to be very low while the IDSA review awarded it the highest rating. The most significant aspects of these trials and their relationship to clinical practice are described here and important details of design and analysis are described below. Two trials, using validated measures for fatigue, demonstrated that antibiotic retreatment with ceftriaxone produced a moderate to large treatment effect for a subset of patients with severe fatigue (Fallon et al., 2008; Krupp et al., 2003). Given that severe fatigue can be profoundly disabling, this is a positive and significant finding. While IV antibiotics are associated with serious risks, the risk may be lower than previously thought. See Table 2 below. Depending on the severity of one’s condition and circumstances, these risks may be acceptable. Therefore, it would be reasonable for physicians to discuss carefully with patients the risk to benefit ratio so as to create a personalized, optimal treatment plan. The failure of any Lyme disease clinical trial to demonstrate treatment efficacy does not necessarily reflect absence of infection, but rather may reflect limitations of the clinical trial design. All of the trials were biased towards treatment failure as none of these studies excluded patients who had already failed to respond to multiple prior courses of antibiotic therapy (Fallon et al., 2008; Klempner et al., 2001; Krupp et al., 2003). The failure to find PCR DNA evidence of Bb in the serum of patients in the antibiotic retreatment trials of Lyme disease is not proof that Bb has been eradicated, as Bb is rarely found in blood even among actively infected patients with new onset untreated Lyme arthritis or new onset untreated neurologic Lyme disease (Aguero-Rosenfeld, Wang, Schwartz, & Wormser, 2005). Table 9. Safety Analysis of NIH-sponsored Trials of Antibiotic Retreatment of Lyme Disease Study N Days of IV (antibiotic/ placebo) Total Days IV device* Sig Adverse Events AE rate/ 1000 IVD Days Klempner 2001 (Klempner et al., 2001) 129 30 3,870 2 (1.6%) 0.5 Krupp 2003 (Krupp et al., 2003) 55 30 1,650 6 (10.9%) 3.6 Fallon 2008 (Fallon et al., 2008) 37 70 2,590 7 (18.9%) 2.7 Total 221 - 8,110 15 (6.8%) 1.8 *For each trial, the total days of IV device (IVD) is based on all subjects completing treatment with their assigned agent/placebo. Given that some subjects did not finish the full course of agent/placebo, the actual number of device days is lower for the individual trials and for the group as a whole than reported here. Therefore, the calculated AE rates per 1000 IVD would be slightly higher than reported here. Study 1 – Fatigue (Krupp et al., 2003) Comments. In this randomized placebo-controlled study of 55 patients with previously treated Lyme disease, all enrolled subjects had to have at least moderate levels of fatigue for entry into this study as assessed by the Fatigue Severity Scale, a well-validated measure of symptom severity; this requirement ensured that all patients would be impaired on the primary outcome measure of interest – fatigue. Of the four NIH-funded studies of post-treatment Lyme symptoms, this study demonstrated the greatest treatment benefit. Sustained improvement in fatigue was observed at six months three times more often among those who received IV ceftriaxone than among those who received IV placebo. There were two other primary outcomes (processing speed and OspA antigen levels in the CSF) that did not change over time, however the authors note in the discussion that these two outcome measures should not be used to assess therapeutic efficacy as too few patients had significant processing speed problems or OspA protein in the CSF at the start of treatment. It is well-known in clinical trials research that a treatment cannot be adequately assessed unless patients show impairment on the domain of interest prior to treatment. Therefore, fatigue was the only scientifically valid primary outcome measure in this study and on the measure of fatigue one month of IV ceftriaxone was shown to be efficacious among patients with Post-treatment Lyme disease. Six patients (10.9%) had a significant adverse event related either to ceftriaxone and/or the intravascular device. Although the therapy was shown to be efficacious, the authors concluded that IV ceftriaxone was not recommended for post-treatment Lyme fatigue, primarily due to the risks associated with IV treatment. Clearly, given the risks associated with IV ceftriaxone therapy, the identification of a safer therapeutic would be beneficial. Given the profound negative impact of living with debilitating persistent fatigue however, the potential benefit of IV ceftriaxone should not be disregarded. Study 2 – Cognitive Impairment (Fallon et al., 2008) Comments. This randomized, placebo-controlled study of post-treatment Lyme encephalopathy showed a mild-moderate improvement in the primary outcome of cognition after 10 weeks of IV ceftriaxone that fell just above the border of statistical significance at the primary outcome time point. Because this improvement in cognition was lost during the drug free interval from 10 weeks to 6 months, 10 weeks of repeated IV ceftriaxone therapy was not recommended for sustained improvement in cognition. Several points should be noted about this trial. First, the enrolled patients were among the most “treatment refractory” as all had persistent symptoms despite previously been treated with oral antibiotics (mean 7 months) and intravenous antibiotic therapy (mean 2 months). Second, the most severe symptoms among the patients were fatigue and pain, not cognition, even though patients were recruited for cognitive impairment; this suggests that future studies of post-treatment Lyme disease should focus on fatigue and/or pain. Third, a small sample size such as the one in this study (37 patients) makes it very hard to demonstrate efficacy. Given these two detrimental factors for a study, it is notable that there was greater improvement with ceftriaxone than with placebo on the secondary outcome measures of pain, fatigue, and physical impairment among the more impaired subgroups when baseline severity was included in the statistical analysis of the entire sample. When this study was compared to the Krupp study of post-treatment Lyme fatigue, similar responder levels were noted (see Figure 4) (Fallon et al., 2012). Image Figure 4. Percentage of patients who reported improvement on fatigue at 6 months after receiving IV CFTX or placebo. Improvement was reported in 64% of patients who received IV CFTX for 1 month and 18.5% of patients who received placebo (P < .001) in Krupp’s study. Improvement was seen in 66.7% of patients who received IV CFTX for 10 weeks and 25% of patients who received placebo (P=.05) in Fallon’s study. However, because these self-report measures were not the primary outcome domains chosen before the study, proof of efficacy requires an independent study to assess these measures as a primary outcome before definitive conclusions can be drawn. Seven patients (18.9%) had a significant adverse event related either to ceftriaxone and/or the intravascular device. Studies 3 and 4 – Functional Status (Klempner et al., 2001) Comments: In this randomized placebo-controlled study of one month of IV ceftriaxone/placebo followed by two months of oral doxycycline/placebo, no difference was noted on the primary outcome measure of mental and functional impairment as assessed by the SF-36. This study was originally designed as two studies (a seropositive group and a seronegative group) using the same study design, but the final analysis pooled both groups together. The study was stopped mid-way after an interim analysis failed to show sufficient evidence of an emerging difference between treatment groups to justify study continuation. This study had the largest sample size (n=129) among the U.S. studies. A significant weakness of this study is that a severity threshold on a validated measure was not used to ensure people were impaired at study entry; although as a whole, the group did have impairment in physical functioning, the range of impairment was wide within this enrolled sample indicating that some of the patients would not have been meaningfully functionally impaired at the start of the study on the primary outcome measure used to assess efficacy (SF-36). If a person is not impaired on the primary outcome measure at entry into the study, then one would not expect any treatment to result in meaningful change. Typically, studies would address this problem by controlling for differences in baseline severity during the data analysis. That analysis was not reported in the published paper. Therefore, because the sample contained a mixed group of patients - those who had mild or little impairment and those who had substantial impairment, the final results of the study are difficult to interpret. In addition, the minimally clinically important difference used to determine success in this trial may have been excessively large thereby limiting the ability of the trial to show a treatment effect (Delong et al., 2012). The PLEASE trial, although not conducted in the United States, is included in this discussion of persistent symptoms because it was published in a U.S. medical journal (Berende et al., 2016) and because it is the largest published study of patients with persistent symptoms after treatment for Lyme disease. Conducted in the Netherlands, this trial addressed whether extended antibiotic therapy is more effective than shorter course therapy for patients with persistent symptoms. This trial was “patient-friendly” in two ways. First, everyone received an initial course of active treatment with 2 weeks of ceftriaxone; the randomization to extended treatment occurred after the first 2 weeks. Second, the trial allowed enrollment of those with well-documented Lyme disease and those who didn’t have well-documented past Lyme disease; by doing this, the investigators addressed one of the limitations of many of the prior clinical trials – lack of generalizability. This trial replicated a clinical situation that is common in physicians’ offices – patients with persistent symptoms whose clinical history is either firm or only probable/possible for past infection. Specifically, patients with persistent symptoms attributed to Lyme disease were included if they had either a) an EM rash that preceded the symptom onset or an otherwise proven case of standard criteria Lyme disease; or b) a positive IgG or IgM Western blot for Lyme disease (without the additional requirement of a preceding positive or equivocal ELISA which is standard for the two-tier algorithm). Patients were all given two weeks of IV ceftriaxone then randomly assigned to 12 weeks of one of the following: doxycycline; clarithromycin and hydroxychloroquine; or placebo. The primary outcome was improvement in physical functioning on the SF-36. The study results revealed that all 3 groups improved over time with no difference between those who got additional antibiotic treatment versus placebo. The conclusion was that longer-term antibiotic treatment did not confer additional benefit in health-related quality of life beyond that obtained with short-term treatment. Ironically, one of the strengths of this study – enhanced generalizability due to broad enrollment criteria – was also one of the weaknesses. It is unclear for example whether patients with a positive IgM Western blot had prior Lyme disease, as IgM Western blots are particularly prone to false positive results. Further, patients with positive IgG Western blots may have had infection that was eradicated by the immune response without ever getting clinical symptoms of disease. In fact, only 96 of the 280 participants (34%) in this trial had objective clinical documentation of prior Lyme disease. Therefore, the negative results of this trial for extended antibiotic therapy cannot be generalized to those patients who have persistent symptoms and a well-documented past history of Lyme disease. Additionally, this trial suggests that there may be a benefit to repeated antibiotic therapy (2 weeks of IV ceftriaxone in this case) as improvement was noted across all 3 groups. However, given the absence of randomization to either ceftriaxone or placebo in the initial 2 weeks of the study, we are left with the question of whether ‘repeated antibiotic therapy’ or the placebo effect or time alone accounted for the improvement in these patients. Educational Gaps/Opportunities Some clinicians may not know that PCR testing of blood and CSF is insensitive and should not be used to guide decisions on whether to retreat. Some clinicians and patients may be unaware that there may be significant differences in antibiotic performance between the “in-vitro” laboratory setting and what happens in infected animals or humans. Research Opportunities/Gaps Identification of markers of active infection in antibiotic-naïve and patients with ongoing symptoms of Lyme disease following antibiotic treatment. One of the major gaps in the clinical care of patients with untreated disseminated disease and those with ongoing symptoms and signs of Lyme disease following antibiotic treatment is the absence of biomarkers of infection. Antibody-based assays, if positive, only confirm that exposure occurred in the past; they do not reveal whether or not infection is currently present. Clinicians are then left with the challenging situation of making a treatment recommendation for or against antibiotics in these patients without knowing whether the infection is still present. A related gap is the insensitivity of the tests for certain manifestations of Lyme disease. For example, two-tier testing is negative 35-50% of the time in early Lyme disease and 10-43% of the time in neuroborreliosis (Aguero-Rosenfeld et al., 2005; Dressler et al., 1993b; Marques, 2015). In the case of the study by Dressler et al, the in-house IgG ELISA was 79% sensitive and the IgG Western blot, using the 5 of 10 interpretation criteria, was 72% sensitive in his well-characterized neuroborreliosis patients, yielding 57% sensitivity for two-tier testing in neuroborreliosis. Reports of newer serologic tests having higher sensitivity may be due to preselection bias as sensitivity is often determined using samples that previously had been positive on two-tier testing. It is important to note that 17% of the subjects included in a 1999 encephalopathy trial were seronegative and their inclusion in the study was based on other parameters (Logigian et al., 1999). In a non-human primate (NHP) study investigating treatment that occurs late in the course of B. burgdorferi infection, the C6 test results dropped to normal over time in all treated animals, regardless of infection status, and in some of the untreated NHPs (Embers et al., 2012). A recent NHP study, documented significant variability in antibody production between NHPs infected with same strain of B. burgdorferi and within individual NHPs over time (Embers et al., 2017). Given that B. burgdorferi expresses different proteins (antigens) over time in vivo and that some of these may not be expressed in vitro (and vice versa), serologic tests based on in vitro antigens sources may not be informative regarding the infection status of humans (Branda et al., 2018). Once a sensitive and specific biomarker of active infection for both early and late manifestations of Lyme disease exists, many of the controversies around the diagnosis and treatment of Lyme disease would be more easily studied and resolved. Identification of biomarkers to clarify pathophysiology of persistent symptoms and to help guide treatment selection. With regard to treatment failures, what is the primary cause of antibiotic failure? If failure is due to insufficient antibiotic levels in the infected tissues, what may account for this? For example, can an individual’s metabolism (or competing/concurrent drugs) diminish the effectiveness of the antibiotic? If persister B. burgdorferi are present and if future research indicates that the recent in vitro findings are applicable to human disease, then other antibiotics or antibiotic approaches might be needed in order to kill not only actively dividing spirochetes but also the more quiescent persister ones. Do combinations of antibiotics confer greater longer-term benefit than single agent regimens? Does B. burgdorferi produce a biofilm? Has the individual developed a post-infectious disorder that now needs treatment with other approaches rather than antibiotics? The majority of patients in the NIH-sponsored retreatment trials had already received more than 1 course of antibiotic treatment for Lyme disease prior to entry into their respective study. study. Thus, the trials were biased toward failure. Effectiveness of retreatment has never been examined in the U.S. among patients who have only had one course of prior therapy. Is the effectiveness of antibiotic retreatment greater in patients who were treated for Lyme Disease with no more than one course of recommended treatment compared to those who received multiple courses of antibiotics? Therapies to Avoid due to an Association with an Increased Risk of Treatment Failure Clinical studies have demonstrated that certain antibiotics and other medications should generally not be used because they either had higher failure rates for treating the infection than commonly used antibiotic agents or were associated with an increased risk of treatment failure. In 1983 study erythromycin was found to be less effective than tetracycline or penicillin for hastening the resolution of the EM rash and alleviating other symptoms of early disease (Steere et al., 1983). Additionally, 14% patients treated with erythromycin experienced disease progression. Cephalexin is ineffective for the treatment of erythema migrans. Of the 11 subjects who initially diagnosed with cellulitis and treated with cephalexin, all have evidence of disease progression (Nowakowski et al., 2000). Not only did the investigators recommend against the use of cephalexin for Lyme disease, they also cautioned that it should not be used to treat apparent cellulitis in Lyme endemic areas during tick season as this would avoid situations where EM lesions that are misdiagnosed as cellulitis receive ineffective treatment. The use of corticosteroids prior to antibiotic therapy increases the risk of antibiotic treatment failure (Bentas, Karch, & Huppertz, 2000; Dattwyler et al., 1988; Jowett, Gaudin, Banks, & Hadlock, 2017). In pediatric patients, the use of intraarticular steroids prior to antibiotic treatment in resulted in more prolonged disease and additional courses of antibiotic therapy (Bentas et al., 2000). A retrospective cohort study of patients with Lyme disease facial palsy (LDFP) demonstrated that corticosteroid use in addition to antibiotics alone or antibiotics and antiviral agents was associated with poorer long-term functional outcomes at 12 months than was seen in patients who only received antibiotics (Jowett et al., 2017). Educational Gaps/Opportunities Clinicians may be unaware that they should avoid prescribing erythromycin for early Lyme disease and reserve its use for situations where patients cannot tolerate doxycycline, amoxicillin, or cefuroxime axetil. Clinicians may be unaware that cephalexin is ineffective and should not be prescribed for Lyme disease Clinicians may be unaware that the treatment of cellulitis in Lyme-endemic areas during tick season should be carried out using antibiotics such as amoxicillin/clavulanic acid or cefuroxime axetil as these agents effectively treat both cellulitis and erythema migrans. Clinicians may not be aware that steroids should not be prescribed for patients with Lyme disease prior to antibiotic therapy. Terminology regarding Lyme disease facial palsy (LDFP) and Bell’s palsy needs to be clarified. Unlike LDFP, the etiology of Bell’s palsy is unknown, often presumed to be viral, and commonly treated with corticosteroids. Interchanging the use of Bell’s palsy for LDFP may mistakenly lead to the inappropriate prescription of corticosteroids to Lyme patients. Question 2: In addition to the gaps/opportunities identified in Question 1, what other gaps/opportunities exist? Innovative research The traditional research model uses randomized controlled trials, which are essential to demonstrate cause and effect. There is a growing recognition that traditional clinical trials are costly, slow, and enroll patients that are unlike those seen in clinical practical. For example, roughly 66% of clinical trials fail to recruit a sufficient number of participants and fewer than 20% of trials meet their deadlines. These delays are estimated to cost over $8 million in lost revenue per day (Andrews, Kostelecky, Spritz, & Franco, 2017). At the same time, there have been calls for less restrictive entry criteria for randomized controlled trials and pragmatic trials that are more reflective of the populations seen in clinical practice. Technological advances available today allow people to connect virtually and have increased our ability to aggregate and share data, increase research efficiency, and accelerate the pace of research exponentially through patient registries, clinical trial consortiums, and analysis of other big data resources. Recognizing the constraints of traditional research and the opportunities afforded by new technological advances, government institutions are adopting big data, patient-centered research, and personalized medicine initiatives, including the PCORI patient-powered network of patient registries and healthcare institutions, PCORnet, President Obama’s Personalized Medicine Initiative, the FDA’s Patient Focused Drug Development program, the Veteran’s Administration’s 1 million veteran’s initiative, the NIH’s big data research program (Etheredege, 2011), the Collaboratory, and the CDC congressionally mandated ALS registry, which includes a patient generated data web portal (CDC, 2018). Pharmaceutical companies are beginning to explore virtual clinical trials—which bring the trial to the patient utilizing home nurses, drug delivery by mail, and online consent and outcomes progress tracking (CenterWatch, 2017). In Lyme disease, these new research approaches hold enormous potential. For example, patient driven registries and research platforms run by patient advocacy organizations, play an integral part of the process, as trusted intermediaries helping to aggregate observational data, fuel hypothesis testing, design patient friendly trials, and aid in the recruitment, analysis, and dissemination of trial results. In the Lyme community, LymeDisease.org launched a patient-powered registry and research platform, MyLymeData, which has enrolled over 10,000 patients. Future research in Lyme disease should include innovative approaches in the way research is conducted, including collaborative approaches between researchers, patients and physicians, new methods of clinical research and data analysis that incorporate modern information technology, and the concept of a pragmatic clinical trials that reflect populations representative of those in clinical care in a rapid learning healthcare system advanced by the National Academy of Medicine ("NIH Collaboratory LIVING TEXTBOOK of Pragmatic Clinical Trials," 2014). Much of what could be learned from the data of thousands of individual patients treated annually that is currently used to fuel research and advance clinical care. Does the presence of other tick-borne diseases increase the risk of treatment failure? In a murine model investigating the effectiveness of prophylactic single-dose doxycycline, when mice were fed on by ticks infected with B. burgdorferi alone, doxycycline was 43% effective for preventing transmission (Zeidner et al., 2004). When mice were fed on by ticks infected with both B. burgdorferi and Anaplasma phagocytophilum the effectiveness of doxycycline for the prevention of B. burgdorferi transmission dropped to 20% (Zeidner et al., 2008). A large survey of Lyme disease patients found that a majority of patients with signs and symptoms following antibiotic treatment report at least one co-infection (see Appendix 8). Thus, the importance of supporting research into treatment outcomes for co-infected patients cannot be understated. Can Research findings from Europe be generalized to United States cases given genospecies and B. burgdorferi strain differences? How does B. burgdorferi escape killing by an intact immune system and/or antibiotic exposure? Does pulse dosing contribute to improved outcome? The evidence is for this treatment strategy is conflicting (Feng et al., 2015; Sharma et al., 2015). Is there a role for immune modulatory therapy, such as IVIG? Summary: Priority 3, The Treatment of Lyme Disease This paper documents that there are still many unknowns in the selection of optimal treatment for various presentations of Lyme disease. . While this review provides additional clarity regarding treatment effectiveness for patients with an erythema migrans rash, it is unable to do so for other clinical presentations as the available evidence is often scant and the quality is low. With regard to later disease manifestations and ongoing symptoms and signs of Lyme disease in patients who received prior antibiotic therapy for Lyme disease, it is clear that new treatment options are needed to address the increasing number of patients with persistent or relapsing symptoms. As new ideas are generated that suggest clinical validity either in animal models, open label human studies, or by hypothesis generation through data mining from large human databases, randomized clinical trials can then be conducted to establish efficacy. Given the massive improvement in basic science technologies, novel clinical trial designs, access to large databases and state-of-the-art biostatistical and data mining techniques, new more effective treatments can be identified in a more rapid and efficient manner. Such multi-level investigations need to be funded to enhance patient care. Development of direct tests of B. burgdorferi infection is critical. Clinical research and patient care is hampered by the lack of biomarkers for active infection. Reliance on serology is prone to error as positive results may reflect prior exposure but not active infection and negative results are not necessarily indicative of the absence of an active B. burgdorferi infection. Additional investigations regarding B. burgdorferi and its ability to establish a persistent infection, the human pathophysiology of Lyme disease, and a wide range of treatment topics are needed. Potential Actions Research Investigate and develop biomarkers that: a) directly identify B. burgdorferi infection in all infected patients, whether they are antibiotic-naïve or previously treated: b) identify subgroups of patients that will benefit from other, non-antibiotic modalities of treatment. Conduct research to identify the biosignature of post-treatment Lyme disease to lead to new ideas for treatment intervention. Precision medicine technologies such as proteomics, metabolomics, genomic, and neuroimaging can provide valuable insight regarding the pathophysiology of persistent symptoms and recovery. Develop innovative patient-centered research approaches that take advantage of technological advances to conduct real-world (pragmatic) trials that reflect the range of patients seen in clinical practice and develop a core set of outcome measures that are patient-centered. Develop a national consortium of investigators and physicians to facilitate Lyme disease clinical trials. This consortium should construct multi-regional trials simultaneously, investigating various agents and treatment durations, using a core set of patient-centered outcomes. Investigate potential causes of treatment failures, such as insufficient antibiotic levels in infected tissues and/or the presence of other tick-borne diseases. Study the extent to which B. burgdorferi alters normal human immune responses and the mechanisms by which B. burgdorferi escapes immune and/or antibiotic destruction. Determine whether persister cells exist in vivo and whether B. burgdorferi produces biofilms. Determine whether repeat antibiotic therapy is more effective in patients who had had one course of antibiotic compared to those who have been treated repeatedly. Educational: Development of comprehensive, peer-reviewed report for clinicians. Peer-review should be done by a group of clinicians and research scientists that represent a wide spectrum of evidence-based medical opinion on these issues. Development of peer-reviewed educational report for patients and the general public. Peer-review should be done by a group of clinicians, research scientists and patients with a broad range of perspectives. Clinician and research scientist stake holders should represent a wide spectrum of evidence-based medical opinion on these issues. Discussion Borrelia burgdorferi, the agent of Lyme disease, is a formidable pathogen able to infect and persist in a number of immunocompetent mammals including humans. Studies in cultured bacteria and animal models have demonstrated that the spirochete accomplishes this persistence utilizing a variety of mechanisms that thwart innate and adaptive immune responses including inhibition of the complement system, suppression of strong inflammatory responses, antigenic variation and suppression of B cell responses. Significant insights have been gained through the use of cultured bacteria and several well-characterized animal models but no single species model reproduces the spectrum of disease seen in humans nor the long-term consequences of neuroborreliosis. Differences between Borrelia burgdorferi sensu lato species and strains and their impact on human disease needs to be explored and much remains to be learned about the spirochete’s ability to survive antibiotic exposure. Additionally, whether Borrelia burgdorferi infections lead to a more generalized suppression of adaptive immunity needs to be explored. If this were the case in humans, those with concurrent tickborne infections may require different diagnostic and treatment approaches. Disease manifestations in humans have been well characterized but host-pathogen interactions and the resultant pathophysiology are poorly understood. The number and severity of these different disease manifestations vary greatly from patient to patient suggesting that host determinants may predispose to more severe or prolonged infection. Long periods of disease latency are not uncommon; what allows for these intervals and what triggers a new episode of disease activity is unknown. B. burgdorferi disseminates widely yet it appears that certain tissue sites are preferred. Factors driving this selectiveness is unknown. Many of the most challenging symptoms and signs that patients contend with are neurological in nature yet whether and how B. burgdorferi is able to establish persistent localization in the nervous system is uncertain. The possibility that Borrelia burgdorferi releases lipoproteins into the circulation that cross the blood brain barrier and a) induce central nervous system inflammation and/or b) directly affect neural tissue needs to be studied. The role of pro-inflammatory cytokines, autoantibodies, and cross-reactive antibodies should be explored. The pathophysiology underlying ongoing symptoms and signs in patients previously treated for Lyme disease is not understood. Potential mechanisms include immune dysfunction, tissue injury, untreated co-infections and persistent B. burgdorferi infection; these mechanisms are not mutually exclusive. All need to be investigated. Establishing highly successful treatment regimens for the various Lyme disease presentations is an ongoing challenge for researchers, clinicians and patients. The current trial evidence is limited in size, scope and quality. While treatment of patients with erythema migrans is usually successful with three weeks of antibiotics, trials have not established whether this should be the minimum duration of therapy. With regard to later disease manifestations and ongoing symptoms and signs of Lyme disease in patients who received prior antibiotic therapy for Lyme disease, it is clear that new treatment options are needed to address the increasing number of patients with persistent or relapsing symptoms. As new ideas are generated that suggest clinical validity either in animal models, open label human studies, or by hypothesis generation through data mining from large human databases, new randomized clinical trials should be conducted to test efficacy. Given the massive improvement in basic science technologies, novel clinical trial designs, access to large databases and state-of-the-art biostatistical and data mining techniques, new more effective treatments can be identified in a more rapid and efficient manner. Such multi-level investigations need to be funded to enhance patient care. Additionally, absent a direct marker of infection, investigators cannot be certain that enrolled subjects are actually infected. This is especially problematic for trials investigating antibiotic retreatment for patients with ongoing signs and symptoms of Lyme disease following antibiotic therapy. Under the current circumstances, determining the most beneficial antibiotic course of action is a difficult endeavor for both clinicians and patients. The subcommittee would like to address an additional topic that is very important to patients and physicians. As discussed in previous sections, Lyme disease is a multi-systema, multi-stage disease, with manifestations that vary and often increase in number and severity the longer the infection goes without antimicrobial intervention. Following initial antibiotic treatment and irrespective of time to diagnosis, many patients remain ill with symptoms of disease that are real, but whose etiology is unknown. The term “Post Treatment Lyme Disease Syndrome” (PTLDS) was coined to study a particular subset of well-characterized patients who have undergone a standard course of antibiotics yet, for unknown reasons, remain ill with similar symptomology to that of the untreated disease state. While this narrower, more specific definition may be useful for some research purposes, its utility in clinical care or diagnosis is limited because it excludes a large portion of the clinical population of patients who have persisting symptoms despite having received some antibiotic therapy. It also limits the generalizability of these studies to the clinical population. In addition, the term “syndrome”, is defined as “a group of signs and symptoms that occur together and characterize a particular abnormality or condition”. No claim of etiology is required in this standard definition. When used in Lyme disease however, PTLDS is often presented as a post-infectious state and the very moniker “Post Treatment Lyme disease” seems to imply to many clinicians, researchers and the public that the possibility of a persistent Borrelia infection is nonexistent. Until the pathophysiology of this illness is known, it seems prudent to use terminology that is inclusive of the varied etiologic hypothesis including persistent infection. The use of the word “syndrome” also has a negative connotation for patients. In the absence of etiology, the term can be misused by doctors to denote a patient who has no objective illness, despite significant symptoms of disease. 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Therapeutic passive vaccination against chronic Lyme disease in mice. Proc Natl Acad Sci U S A, 94(23), 12533-12538. Zimmer, G., Schaible, U. E., Kramer, M. D., Mall, G., Museteanu, C., & Simon, M. M. (1990). Lyme carditis in immunodeficient mice during experimental infection of Borrelia burgdorferi. Virchows Arch A Pathol Anat Histopathol, 417(2), 129-135. Appendix 1. Mechanisms of invasion by Borrelia burgdorferi Many pathogens that invade hosts utilize surface exposed or secreted proteases to mediate this effect. Borrelia burgdorferi is unique in that it appears to make no known secreted protease, although a surface exposed protease, designated HtrA, has been identified (Backert et al., 2018; Russell et al., 2013; Ye et al., 2016). One mechanism that uses host proteins involves the interaction with mammalian plasminogen, which is bound to the outer surface of B. burgdorferi and then activated by host urokinase (Coleman et al., 1997; Hu et al., 1995; Klempner et al., 1995, 1996). Activated plasmin then can degrade the extracellular matrix, which promotes assisted invasion into host tissues (Coleman et al., 1995; Klempner et al., 1993) Appendix 2. Genes involved in dissemination within the vertebrate host After colonization, B. burgdorferi replicates at sites of adherence and then spreads (disseminates) into deeper tissue (Radolf et al., 2012). The spirochetes are actively motile at this point and use this attribute to invade deeper tissues and “out run” innate immune cells. B. burgdorferi makes no known toxins and is not known to secrete proteins (e.g., proteases) that mediate the breakdown of host structures (Casjens et al., 2000; Fraser et al., 1997). To circumvent this limitation, the spirochete coats itself with host proteins that can substitute for this effect (see Appendix 1 for more detail). This “hijacking” of host proteins allows B. burgdorferi to invade and spread to ultimately find sites that are protected from clearance via the immune response. Invasion into connective tissues results in secondary colonization within lymph node, joint, cardiac, spleen, and bladder tissues, as well as skin sites remote from the initial infection location. Elegant confocal microscopy that track fluorescent B. burgdorferi in mice demonstrated that B. burgdorferi bind to blood vessels in a manner that can withstand the force of blood moving over them (for example., shear stress) (Ebady et al., 2016; Moriarty et al., 2012). In some rare instances, spread through the blood vessels into deeper tissues has been observed (Kumar et al., 2015; Moriarty et al., 2008; Norman et al., 2008). Additional studies showed that material derived from Lyme spirochetes can persist within murine connective tissue and may serve to provide a site for continued inflammation (Bockenstedt et al., 2012). Despite these significant advances, we still do not understand much molecular detail regarding how B. burgdorferi disseminates. Although initially presumed to be mediated hematogenously, based on the activation of host proteolytic systems and phenotypic evidence via in vivo imaging (Bockenstedt et al., 2012; Kumar et al., 2015; Moriarty et al., 2008), suggest that spread may also occur through the lymphatics and the interstitium directly. Additional studies are warranted to further evaluate these possibilities. Appendix 3. Evidence of B. burgdorferi Persistence in Patients Who Remained Ill 6 or More Months Post-Antibiotic Treatment for Lyme Disease Positive (+) Culture Haupl T – 1993: (+) culture, PCR, electronmicroscopy; Hudson BJ – 1998: (+) culture of skin biopsy with (+) direct immunofluorescence antibody staining; Oksi, J – 1999: (+) culture, blood 1/9; (+) PCR 8/9; Pfister HW – 1991: (+) CSF culture; Preac-Mursic V – 1989: (+) culture from CSF; Preac-Mursic V – 1993: Case 5: (+) CSF culture Positive (+) Polymerase Chain Reaction Battafarano – 1993: (+) PCR, silver stain; Cassarino DS – 2003: (+) PCR; Chancellor MB – 1993: (+) silver stain with immunohistochemical staining; Coyle P – 1995: Case 4: (+) OspA antigen CSF; Case 12: (+) OspA antigen CSF; Fraser DD – 1992: (+) PCR of leukocytes; Frey M – 1998: (+) PCR of muscle biopsies; Lawrence C – 1995: (+) PCR of spinal fluid; Liegner K –: (+) PCR of whole blood x2 and PCR skin L thigh lesion Warthin-Starry on bx; Nocton JJ – 1994: (+) PCR of joint fluid; Nocton JJ – 1996: (+) PCR of CSF; Oksi J –2007: patient 1: PCR+ skin for B. burgdorferi; Tager FA 2001– : (+) whole blood PCR Positive (+) Microscopy Cimmino MA– 1989: Spirochetes histologically demonstrated in spleen after IV PCN G; Kobayashi K – 1997: Warthin starry (+) brain tissue at necropsy with (+) serology; Waniek C – 1995: (+) Dieterle stain for spirochete in pt with (+) serology for Lyme and (-) VDRL Appendix 4. Data from Published Patient Surveys and MyLymeData Compared to other chronic illnesses, patients with ongoing symptoms and signs of Lyme disease following antibiotic treatment are more likely to rate their health as fair or poor (Johnson et al., 2011). Image *CD refers to Cardiothoracic Disease **PTSD (vets only) refers to Post Traumatic Stress Disorder among military veterans Figure 5. Percentage of survey respondents reporting fair or poor health compared to the general population and patients with other chronic diseases were as follows: 72% for chronic Lyme disease (n= 3021), 62% for congestive heart failure (n=2), 59% for fibromyalgia (n=3), 54% for cardiovascular disease: stroke (n=1), 51% for cardiovascular disease: heart attack (n=1), 47% for post-traumatic stress disorder among military veterans (n=4), 46% for diabetes (n=5), 43% for systemic lupus (n=6), 37% for multiple sclerosis (n=7), 34% for irritable bowel syndrome (n=8), 32% for liver failure (n=9), 32% for depression (n=10), 31% for arthritis (n=5), 31% for asthma (n=1), 29% for hypertension. (DOI: https://peerj.com/articles/322/#fig-1) Appendix 5. Reduction in health status may be reflected in the fact that many patients with ongoing symptoms and signs of Lyme disease following antibiotic treatment altered their work status. Image Figure 6. Change in Work Status for Patients with Ongoing Symptoms and Signs Following Antibiotic. Of 100 percent of the patients, 15% made no changes to full-time work, 3% made no changes to part-time work, 7% changed nature of full-time job, 3% changed nature of part-time job, 6% reduced full-time work, 10% reduced part-time work, 43% stopped working, 13% were not previously employed. (DOI: https://peerj.com/articles/322/#fig-5) Appendix 6. Patients value shared decision-making Image Figure 7. Factors patients with Lyme disease identified as important in making medical decisions and the respective response rates: possibility of preventing disease progression (73%); my level of functional impairment (70%); severity of my illness (70%); availability of alternative treatments (52%); whether treatment beneficial in past (46%); ability to tolerate side effects (45%); cost of treatment (44%) Appendix 7. Most patients had late stage disease when they were diagnosed with Lyme disease. Table 10. Stage of Illness – Current vs at Time of diagnosis Stage of Illness Current Stage Diagnosis Stage Chronic LD (remained ill for at least 6 months after treatment w antibiotics for 10-21 days) 61% - Late stage untreated LD (6 months after symptom onset) 18% 70% Early or disseminated LD (flu-like, neurologic, cardiovascular or musculoskeletal symptoms) 5% 16% Initial Erythema migrans (EM) bull’s eye or irregular rash 1% 5% Tick bite occurred but no symptoms 0 1% Other 11% 5% Don’t know 4% 4% Appendix 8. Co-infections are a common feature in patients with Lyme disease Image Figure 8: Co-infection Rates in Patients with Lyme Disease –Babesia: 23% with positive test, 21% without positive test; Bartonella: 19% with positive test, 23% without positive test; Mycoplasma: 15% with positive test, 4% without positive test; Ehrlichia/Anaplasma, 11% with positive test, 5% without positive test; Rocky Mountain spotted fever: 5% with positive test, 2% without positive test. Appendix 9. Subcommittee Co-Chairs, Members, Staff, and Non-member Supporter Working Group and Subcommittee Members John Aucott, MD Working Group Chair, Associate Professor of Medicine, Director of the Johns Hopkins Lyme Disease Clinical Research Center Wendy Adams, MBA Subcommittee Co-Chair, Bay Area Lyme Foundation Estella Jones, DVM Subcommittee Co-Chair, Acting Deputy Director, Office of Counterterrorism and Emerging Threats, FDA David A. Leiby, PhD, Chief, Product Review Branch, Center for Biologicals Evaluation and Research (CBER), FDA James Berger Alternate Designated Federal Officer, Office of HIV/AIDS and Infectious Disease Policy, U.S. Department of Health and Human Services Nicole Baumgarth, DVM, PhD, Professor, Center for Comparative Medicine and Department of Pathology, Microbiology & Immunology, UC Davis Pat K. Coyle, MD, Neurology Professor and Vice Chair, Department of Neurology, Director, MS Comprehensive Care Center, Stony Brook University Medical Center Sam Donta, MD, Professor of Medicine (retired), Fellow Infectious Diseases, Infectious Disease Society of America; Consultant, Infectious Diseases Brian Fallon, MD, MPH, Professor of Clinical Psychiatry, Director, Lyme and Tick-Borne Diseases Research Center, Columbia University Lorraine Johnson, JD, MBA, CEO, LymeDisease.org Elizabeth Maloney, MD, President, Partnership for Tick-Borne Diseases Education Jon Skare, PhD, Professor and Associate Head, Department of Microbial Pathogenesis and Immunology, Texas A & M University Brian Stevenson, PhD, Professor, Department of Microbiology, Immunology & Molecular Genetics, University of Kentucky College of Medicine Non-Member Coordination and Support Yanni Wang, PhD, (contractor), Kauffman and Associates, Inc. Appendix 10. Subcommittee Agendas and Top-Line Meeting 1, February 26, 2018 Agenda Overview of process: additional information SharePoint: a place where living documents will be stored once the site is set up Mechanics of meeting set-up: new vendor and required timeline for documents to be presented Finalize the complete/full list of key issues and vote Finalize the prioritized list and vote Go over timeline for work and report; set up timeframes for presentations and discussions, plus writing Summary The subcommittee briefly reviewed the work process and mechanisms for future meeting setup, as well as how to share documents through SharePoint. The subcommittee devoted the rest of the conference call to discussing and finalizing a full list of key issues related to pathogenesis, transmission, and treatment of tick-borne diseases. Meeting 2, March 5, 2018 Agenda Presentation on Borrelia infection and the effect on immune system in mouse models Presentation on B. burgdorferi persistence in animal models Q&A Group discussion Summary Subcommittee member Nicole Baumgarth, DVM, PhD, provided an overview of Borrelia burgdorferi infection and its effects on the immune system. Invited speaker Monica E. Embers, PhD, provided an overview on human Lyme disease, treatment regimes, and antibiotic efficacy. She also reported findings of her group’s studies in non-human primates (NHP). Led by Co-Chair Wendy Adams, the subcommittee discussed what they had learned from the two presentations. Wendy reminded the group to keep in mind the priorities the subcommittee had identified. Meeting 3, March 12, 2018 Agenda Presentation 1 Presentation 2 Q&A Meeting adjournment Summary Subcommittee member Jon Share, PhD, provided an overview of pathogenesis-related features of B. burgdorferi, particularly the interactions between B. burgdorferi and its hosts, the role of gene regulation in B. burgdorferi transmission and colonization, and the subversion of the innate immune response. Subcommittee member Brian Stevenson, PhD, provided an overview of studies on how B. burgdorferi recognizes its environment and then responds accordingly. He explained that his research approach to B. burgdorferi and Lyme disease is to discover molecular mechanisms of the bacteria and the disease. Following the presentations, the group discussed a range of issues, including protein functions, symptomatology and the effect of pH on the growth of B. burgdorferi, changes in the shape B. burgdorferi depending on the environment, as well as controversies in the field. Meeting 4, March 19, 2018 Agenda Discussion and vote on the Background section Discussion of the outline for priority 1 Priority 2 and invited presentation Meeting adjournment Summary The subcommittee reviewed the Background Section they had developed prior to the conference call, and discussed the outline for Priority 1 as well as topics related to Priority 2. Subcommittee member Sam T. Donta, MD, gave a presentation based on his research and clinical experience treating patients with Lyme disease. He pointed out a list of issues related to pathophysiology, including initial infection, symptom causation, and mechanisms of persistence. He also shared lessons learned from antibiotic treatment regimens. Meeting 5, March 26, 2018 Agenda Discussion of progress and timeline Discussion of pathophysiology of humans and outline Invited presentation on Borrelia infection in animal models Meeting adjournment Summary The subcommittee discussed the process of developing the final report to working group. Co-Chair Wendy Adams encouraged the subcommittee to 1) use tables and graphs to effectively convey information, 2) focus on clinical data resulted from the U.S., and 3) provide comprehensive literature review and data analysis. Meeting 6, April 2, 2018 Agenda Housekeeping items Status report Discussion of priority 2 Meeting adjournment Summary Co-Chair Wendy Adams briefly explained a couple housekeeping times. Subcommittee members Nicole Baumgarth and Betty Maloney provided quick updates on the priorities their sub-writing groups have been working on. The subcommittee devoted the rest of the conference call to discussing the outline for Priority 3 that Wendy put together prior to the conference call. Meeting 7, April 9, 2018 Agenda Invited presentation on evidence-based medicine and GRADE Invited presentation on Immune mechanisms in post-treatment Lyme disease syndrome Discussion Meeting adjournment Summary Betty Maloney, MD, provided a brief review of evidence-based medicine. Armin Alaedini, PhD, provided an overview of his more than 50 years of research on Post-Treatment Lyme Disease Syndrome (PTLDS). The subcommittee briefly discussed the status of Priority 1 and Priority 2. Meeting 8, April 16, 2018 Agenda Presentation 1: Optimal Antibiotic Regimens for Acute and Persistent Lyme Disease Presentation 2: Patient Centered Healthcare, Trial Innovation, and Early Results from MyLymeData Discussion and review of P3 outline, P1 report status, and P2 write-up. Summary Ying provided an overview of his research on Lyme disease with a focus on persistent phenomena and better treatment for Post-Treatment Lyme Disease Syndrome (PTLDS). Lorraine briefly reviewed the history of patient-centered health care. The subcommittee briefly discussed the statuses of priority 1, 2, and 3. Meeting 9, April 23, 2018 Agenda Presentation: A brief review of the U.S. clinical trials Discussion of draft P2 and P3 outline Vote on P3 outline Logistics Summary Brian provided an overview of five randomized, placebo-controlled clinical trials on Lyme disease that were conducted in the U.S. and Netherlands. The subcommittee briefly discussed the statuses of priorities 2 and 3, voted on the P3 outline, and made plans to finalize and vote on the final reports in the following days. Meeting 10, April 25, 2018 Agenda Discussion of and vote on P1 Discussion of draft P2 and P3 outline Work on P3 Summary The group discussed sections of the report and outlined remaining responsibilities that needed to be concluded before finishing the report. The group also voted on the Potential Actions to be included in the report. Meeting 11, April 30, 2018 Agenda Discussion of priority 2 Discussion of priority 3 Priority 1 status update Discussion of slide presentation Summary The subcommittee reviewed and discussed the current version of the Results section for Priorities 2 and 3. They also discussed how to organize the slides. Meeting 12, May 2, 2018 Agenda Status reports for P2 and P3 Discussion of slide presentation Next step Summary The group discussed the slide presentation to the Working Group and reviewed logistics and timelines for finishing the report.