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ILADS > ILADS Conferences > ILADS Annual Conference 2020 > ILADS 2020 Annual Conference – Abstract

Abstract List

A One Health Approach in the Time of COVID

Juergen A. Richt, DVM, PhD

One Health is a collaborative, multisectoral, and transdisciplinary approach, working at the local, regional, national, and global levels, with the goal of achieving optimal health outcomes for people, animals, and plants by recognizing the interconnection between them and their shared environment. The One Health approach involves all levels of academia, government, industry, policy and research. Some of the global issues One Health works to address include environmental contamination, habitat use, biodiversity loss, antimicrobial resistance, ecosystem function, and emerging infectious diseases. Nearly 75 percent of emerging human infectious diseases in the past three decades originated in animals and since the same microbes infect animals and humans, efforts by just one sector cannot prevent or eliminate the problem. In order to effectively contain them, a well-coordinated approach in humans and in animals is required. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19), an emerging respiratory infection caused by introduction of a novel coronavirus into humans in late 2019 in China. As of June 30, 2020, SARS-CoV-2 has spread to more than 215 countries, has infected more than 10 million people and has caused more than 500,000 deaths. SARS-CoV-2 is thought to be transmitted from an animal reservoir to humans, with recent genetic analyses pointing to bat coronaviruses as evolutionary precursors of SARS-CoV-2. Since humans do not have pre-existing immunity against SARS-CoV-2, it is urgent to develop therapeutics and vaccines to mitigate the current pandemic and to prevent the re-emergence of SARS-CoV-2 in the future. This presentation summarizes the establishment of preclinical animal models for COVID-19 to test vaccine candidates and therapeutics. The One Health approach to COVID-19 research will result in more information sharing related to disease detection, diagnosis, and research, specifically in the development of new therapies and vaccines.

Bartonella Associated Cutaneous Lesions

Edward B Breitschwerdt, BS, DVM

Background: Bartonella species are globally important emerging pathogens not known to infect animals or humans in North America prior to the HIV epidemic. Cat Scratch Disease, an acute Bartonella henselae infection, is associated with a spectrum of dermatological diagnoses. Bartonella Associated Cutaneous Lesions (BACL) also occur in association with chronic intravascular infection. Ongoing improvements in diagnostic testing modalities have allowed discovery of Bartonella spp. DNA in blood, cerebrospinal fluid, and skin of patients with cutaneous lesions, fatigue, myalgia, and neurological symptoms.

Aims: We describe Bartonella spp. testing in participants reporting neuropsychiatric symptoms, the majority of whom reported concurrent cutaneous lesions.

Patients and Methods: Study participants completed a medical history and risk factor questionnaire and provided cutaneous lesion photographs. Study reviewed and approved by NCSU IRB; approval #1960. Bartonella spp. serology and Bartonella alpha proteobacteria enrichment blood culture/ddPCR (BAPGM/ddPCR) were assessed.

Results: Between August 2018 and October 2019, 33 participants enrolled; 29/33 had serological and/or PCR evidence supporting Bartonella spp. infection, of whom 24 (83%) reported concurrent cutaneous lesions since illness onset. Only 10/24 participants reported consulting a dermatologist, whereas 18/29 and 21/29 reported consultation with a neurologist or psychiatrist, respectively. Participants reported median illness durations of 57.7 months (range 4 months to 13 years).

Sixteen participants were both PCR positive and IFA seroreactive to one or more Bartonella spp. antigens. Seven participants were Bartonella spp. seroreactive but PCR negative, and six participants were PCR positive but not seroreactive. Overall, 22 participants were PCR positive, 4 by qPCR and 22 by ddPCR. Of the 22/29 ddPCR positive participants, 2 were positive only from blood, 13 were positive only after BAPGM enrichment culture (7, 14, or 21 days), and 7 were positive from both blood and BAPGM enrichment culture.

Discussion: Cutaneous lesions were common among people reporting neuropsychiatric symptoms and Bartonella spp. infection. To avoid false negative associations, physicians should consider advanced microbiological Bartonella spp. testing procedures in patients with neuropsychiatric symptoms. Studies are needed to determine if, or to what extent, Bartonella spp.might contribute to cutaneous lesions in patients with neuropsychiatric symptoms.

Visualizing the Intersection of Lyme and COVID: Lessons to be Learned

Marna E Ericson, PhD

Background: Acute or persistent, co-infection with more than one pathogen represents a significant, unmet diagnostic and treatment challenge. Patients who are exposed to vector-borne pathogens typically harbor more than a single pathogen.

Aims: Our objective is to develop new diagnostic protocols, using advanced imaging techniques coupled with forward-thinking molecular analyses to accurately and consistently detect and monitor vector- borne infections. We will develop novel diagnostic protocols that will confirm infection (acute and persistent) and importantly, examine the pathogenesis of infection. Coupled with our focus on vector-borne infections we are applying this same multi-pronged approach to COVID. The intersection of “Lyme” and COVID are discussed.

Patients and Methods: Patients (n=30) will be consented and enrolled using an IRB-approved protocol. Subjects must be 18+years old and have a one-year history of Lyme-associated symptoms. Skin biopsies and blood is collected, de-identified and processed. Both RNA and DNA, extracted from blood and skin, will be analyzed by PCR, a target-based approach and with the MinION, an unbiased approach that can reveal co-infection status of patients. Both blood and skin will be processed and stained using a combination of biomarkers including RNA-fluorescence in situ hybridization, antibody labeling, direct-binding of select fluorescent-labeled lectins and nuclear dyes. Processed samples will be analyzed using a suite of advanced imaging protocols including single- and multi-photon microscopy

Results: Patient recruitment and preliminary sample analysis will be discussed. Using our imaging techniques, multi-labeled blood and skin images reveal a population of bacteria in the dermis both within the vasculature and in the dermis, associated with dermal collagen. Association with the peripheral nervous system and lymphatics will also be presented. Data using the RNA-FISH probes on skin and blood will also be presented both for Bartonella spp. infection and COVID.

Discussion: Our broad-based analyses of samples from Lyme patients will provide a more comprehensive understanding of the complexity of vector-borne disease pathology. Indeed the role of the dermal niche and the interplay of multiple pathogens coupled with the host response, e.g. cytokine storm, may provide information to more effectively guide treatment regimens. In the future we hope to follow these patients through and after their treatment. The increased appreciation of the host response, along with the data on how disease pathogenesis affects the vascular, lymphatic and nervous system provides invaluable data for understanding response, persistence and resolution of vector-borne disease as well as COVID.

COVID-19 Confirmed What We Already Knew from Lyme Disease

Robert Bransfield, MD

Although Lyme disease and COVID-19 are caused by different pathogens with different transmission, the approach to COVID was a repetition of experiences with Lyme disease. We saw the following: healthcare policies often fail with emerging diseases; CDC disease statistics, antibody testing, and case definitions can be misleading; many disease diagnostic and treatment guidelines are flawed; peer reviewed articles in prestigious journals can be fraudulent; flawed randomized controlled trials can create a fool’s gold standard; politicians make catastrophic, fatal, errors with healthcare policies; money and politics corrupt science and medicine; and treating patients cannot be delayed waiting for a vaccine. Emerging diseases can best be overcome by recognizing we can learn the most from our patients, community doctors who treat patients know more than bureaucrats, we need to give significant consideration to the clinical judgment of front line physicians, and chronic and relapsing infection occurs and must be recognized.

The Molecular Toolbox Necessary for the Analysis of COVID-19 and Lyme and Associated Diseases

Lynne Bemis, PhD

Molecular methods applied to patient samples will be described and compared between COVID-19 and Lyme and associated diseases.

The objective of this project is to identify novel molecular approaches to study both SARS-CoV-2 and Lyme and its co-infections. An additional aim is to identify noncoding RNAs from the host that may potentially regulate the cytokine storm or be utilized as biomarkers for predicting how patients are responding.

A literature review was conducted to identify novel molecular methods used to identify SARS-CoV-2 in patient samples. Nanopore technology is used to identify as a key method for defining the mutations associated with the SARS-CoV-2 genome. We have previously utilized this technology to study Bartonella spp. in patient samples. Publicly available sequences were analyzed and provided the data to propose potential molecular mechanisms regulating the virus. These same methods have been utilized to understand the host response in Lyme and associated infections.

Molecular methods were used to identify patients with current SARS-CoV-2 infection, to monitor the mutations in the virus as it evolves and to investigate the host response during the cytokine storm. Studies strongly support similarities between the cytokine storm associated with COVID-19 and that observed in Lyme and associated disease. Similarities in the molecular regulation of the cytokine storm are the focus of these studies. Novel molecular methods will be discussed.

We hypothesized that methods currently in use to study Lyme and associated infections could also be applied to SARS-CoV-2. The outcome of the literature review revealed numerous studies applying the Nanopore technology (deep sequencing), to study infectious agents in patient samples. An advantage of this technology is that it is readily usable in many laboratories worldwide and it provides longer reads needed to distinguish variants. We have utilized this technology to detect Lyme and associated infections as well. An additional outcome of this study is to identify mechanistic connections between the host response and the infection whether viral or bacterial. Thus, similarities in gene expression during the cytokine storm were identified and compared between COVID-19 and Lyme associated infections.

Lyme Disease: Acute and Chronic-Defined

Samuel M Shor, MD

Objective: Chronic Lyme disease has been a poorly defined term and often dismissed as a fictitious entity. In this paper, the International Lyme and Associated Diseases Society (ILADS) provides its evidence-based definition of chronic Lyme disease.

Definition: ILADS defines chronic Lyme disease (CLD) as a multisystem illness with a wide range of symptoms and/or signs that are either continuously or intermittently present for a minimum of six months. The illness is the result of an active and ongoing infection by any of several pathogenic members of the Borrelia burgdorferi sensu lato complex (Bbsl). The infection has variable latency periods and signs and symptoms may wax, wane and migrate. CLD has two subcategories, CLD, untreated (CLD-U) and CLD, previously treated (CLD-PT). The latter requires that CLD manifestations persist or recur following treatment and are present continuously or in a relapsing/remitting pattern for a duration of six months or more.

Methods: Systematic review of over 250 peer reviewed papers in the international literature to characterize the clinical spectrum of CLD-U and CLD-PT.

Conclusion: This evidence-based definition of chronic Lyme disease clarifies the term’s meaning and the literature review validates that chronic and ongoing Bbsl infections can result in chronic disease. Use of this CLD definition will promote a better understanding of the infection and facilitate future research of this infection

Disulfiram In The Treatment Of Lyme & Babesiosis - Retrospective Review Of First 3 Years' Experience In One Medical Practice

Kenneth B Liegner, MD

High throughput screening found disulfiram more potent versus Bb than conventionally recommended antibiotics in vitro(1). This prompted off-label use of this agent as previously reported (2).

We report our subsequent experience with disulfiram between 3-15-2017 and 3-15-2020.

Patients were evaluated in the ordinary course of clinical care. In addition to standard treatment methods disulfiram was mentioned as a possible option for the treatment of Lyme disease. For those preferring disulfiram, full discussion was held regarding potential risks, potential benefits and considerable uncertainties with novel application of this agent. 4 of 71 patients were 'lost to follow-up' resulting in 67 evaluable patients.

Of 67 evaluable patients:
13 of 33 patients (39%) who completed one or two courses of high-dose disulfiram (> or = 4 mg/kg/day) were able to enjoy 'enduring remission' defined as remaining clinically well for > or = 6 months without further anti-infective treatment.
62 of 67 patients (92.5%) endorsed net benefit from the received course or courses of disulfiram, combining the low dose group (<4mg/kg/day), the high dose group and the 'cross-over' group.
5 of 67 patients (7.5%) reported either no benefit or unclear benefit from application of disulfiram
It is unclear whether a low dose regimen can yield 'enduring remission' after discontinuation of treatment
10 of 67 patients (15%) reported development of paresthesias thought consistent with disulfiram-induced peripheral neuropathy. These symptoms completely or substantially resolved over weeks to months with only minimal if any residua in 7 of 10. Mild to moderate residual persisted in 3 of 10.
21 of 67 patients (31%) exhibited emotional instability ranging from hypomania to anxiety and/or depression. Mood disturbance resolved within days to weeks in all patients with cessation of disulfiram although a few required psychiatric intervention.
10 of 67 patients (15%) exhibited mild to moderate hepatic transaminitis which required cessation of treatment in 2 patients. Transaminitis fully resolved in all cases.

Disulfiram monotherapy is useful in the treatment of Lyme disease. Regular laboratory monitoring and close clinical follow-up is necessary. Dosages of 4-5 mg/kg/day for 6-12 weeks appear to be optimal for attempting to achieve 'enduring remission' while minimizing adverse effects. Dosages as low as 0.06 - 2 mg/kg/day for indeterminate durations also conferred benefit with minimal adverse effects. An individualized and flexible approach with shared decision-making is particularly suitable in the use of this agent

Avoiding Cytokine Storm: Controlling Inflammation and Hypercoagulation in COVID

Kristine Gedroic, MD

Discuss the scientific literature on the known mechanism of infection of SARS-CoV-2, the role of Zinc and the concept of an ionophore, particularly chloroquine. Further discuss the phasic nature of the SAR CoV2 and how the second stage is met with complications due to cytokine storm and hypercoagulation. Discuss management strategies to avoid severe outcomes and regulate inflammation. Touch on the application of these concepts to tick-borne disease and post-Lyme sequelae.

Mining for Lyme: Novel Informatics-Driven Frameworks to Study Disease

Rob J. Kulathinal, PhD

Complex diseases that are modulated by multiple unknown genetic, social, and environmental factors present a challenge to researchers. New approaches that leverage the power of big data and informatics may hold the key in providing novel and unparalleled insight into human disease including those conditions which remain particularly elusive. In this talk, I will describe several new frameworks that draw upon the dense, rich, and nuanced interactions between an individual and their biological (One Health) and social (Biosocial) environments as well as the evolutionary history of certain diseases (Evolutionary Medicine). By applying methods such as network analyses, geographical clustering, and phylogenomics on heterogenous and often unstructured data found in our rapidly expanding public and private databases, these emerging frameworks may each be able to provide a powerful, inexpensive, and real-time frontline in our battle against debilitating human conditions such as Lyme Disease.

Specific PCR Phages Tests Show Borrelia miyamotoï More Realistic Prevalence in Europe. COVID/LD Co-Infection Markers and Shared Treatments

Louis Teulieres, MD, PhD

Specific PCR phages tests show Borrelia miyamotoï more realistic prevalence in Europe . Covid /LD co infection markers and shared treatments.

Phages are specific viruses. Inserting their genetic material in their host, they can either produce numerous copies that further destroy the bacteria, or stay in a dormant lysogenic stage and be expressed under specific circumstances.
Phelix Charity and Leicester University developed a Borrelia SL and a specific B. miyamotoi Phage PCR tests already validated in early and late stage patients and healthy volunteers ,showing >90% sensitivity and 100% specificity.

Phages being an evidence of bacterial activity, this technology is an in-vivo amplification system for active infections. Clinicians can evaluate their treatments and discriminate the active disease from the post-lyme syndrome. Borrelia phage based PCR experience is being used for Bartonella spp., Rickettsia spp tests’ developments , and testing new anti infectious compounds and strategies

Borrelia SL and a specific B. miyamotoi Phages PCR Tests were performed on2200 samples (all late stage) from various European countries and from USA All results were verified by sequencin

30 % of the 2200 samples were negative , 70% positive among which 60 % Borrelia miyamotoi .
Interestingly, 20 positive patients were also early stage symptomatic COVID -19 cases ( anosmia ;cough and dyspnea; fever ) Immunological and hematological evaluation put in evidence Th1/TH2 imbalance , D dimers increase , Low IL10, high expression of IL 1 andIL-6 .Successful treatment was 7/ 8 days with macrolids (clarithromycin 500mg X2) , atovaquone 200mg (or indomethacin : 3 x25 mg ) /day .

This is the first large scale report on prevalence of B. miyamotoi in late stages of borreliosis in Europe Results were permitted by the high sensitivity of Phages PCR tests. B miyamotoi resistance to antibiotics and predominant information exchange via plasmids may explain the greater prevalence of miyamotoï type persisters.

Tick biting season should provide genetic typing in early stages, (before antibody production) that will be presented. Early sequential treatment may be helpful to limit serious cases of COVID-19 incidence and tackle morbidities due to LD/COVID coinfections

Lyme Disease – Are We Looking for a Wrong Culprit?

Tatjana Mijatovic, PhD

Background: Borrelia-related diseases (Lyme disease and relapsing fevers) are increasingly prevalent, severe, difficult to diagnose and treat. The high failure rate of tick-borne infection testing undermine treatments‘ strategy and monitoring.
Aims: The goal of this contribution is to bring the focus on the importance to enlarge borreliosis-related testing targets and shed some light on high prevalence of B. miyamotoi presence both in ticks and late stage undiagnosed patients.

Methods: Bacteriophages could become a diagnostic tool based on the principle that if there are phages it is because there are living bacteria. Phelix Charity together with Leicester University microbiology department have recently developed a Borrelia Phage-based PCR test searching for 3 major Borrelia groups (Borrelia burgdorferi sl (including B. burgdorferi ss, B. afzelii, B. garinii, B. spielmanii, etc), Borrelia miyamotoi and Relapsing fever group (B. recurrentis, B. hermsii, etc). This method is efficiently used to assess both human samples and ticks.

Results: Since July 2019, over 2100 results from patients originating various countries have been obtained. Testing included mainly late stage / chronic patients and the aggregated data are showing 30 % negative results and 70% positive among which over 60 % indicated the presence of specific Borrelia miyamotoi phages. Furthermore, ticks from 2019 and 2020 have been analyzed by the same method. The obtained results on ticks showed that over 60% were found positive for Borrelia miyamotoi and only 15% for B. burgdorferi sl.

Conclusion: This is the first large scale report on prevalence of B. miyamotoi in the ticks, as well as in late stages of borreliosis. Seen a high prevalence of B. miyamotoi in tested ticks, further supported by similar percentages found in tested patients, one can hypothesize that the high failure rate of current two-tier screening testing, searching for B. burgdorferi sl only, might be due to the wrong testing target. In other words, the overall high expansion of undiagnosed Lyme disease cases worldwide might be linked to the screening choice focusing only on B. burgdorferi sl and only rarely testing for B. miyamotoi while the later one seems to be much more prevalent. Further accumulation of data both from the patients and ticks should bring the answer to the question are we searching for a wrong culprit. Searching for actual bacterial presence using phage-based testing might pacify the debate and controversies on testing choices and late/chronic stage patients.

Global Species Level Characterization of Tick-Borne Diseases within Ixodes pacificus via MCSMRT Full Length 16s rRNA Microbiome Analysis

Kayla M Socarras, MS

Introduction: In the past century, Tick-borne diseases (TBD) have risen to unprecedented levels. The TBD vector, Metastigmata ticks, feed throughout several life cycles and exchange with their prey a compliment of microbes within the tick microbiome. Despite awareness that some TBD pathogens are transmitted during this exchange, it is poorly understood how all the microbes within the tick microbiome impact human morbidity and mortality.

Aims: This lack of understanding is due, to the use of microbiome assays with insufficient bacterial species resolution. Our aim is to obtain a comprehensive species-level characterization of the tick microbiome as well as data specifying the differences among tick species, and individual tick variation - particularly with respect to the geographic site of collection. These investigations were designed to determine the ratios/percentages of known and suspected pathogens; the percentage of ticks carrying multiple pathogens; and the differences among tick populations in different geo-locales. Acquisition of such high-fidelity microbiome data will provide critical geo-locale defined insights to manage patients suffering from TBD(s).

Methods: The composition of the western black legged tick, Ixodes pacificus, microbiome from northern California was determined using a high fidelity, pan-domain, species-specific, full length 16S rRNA amplification and long-read sequencing protocol followed by bioinformatic analyses using the McSMRT data analysis and database, originally developed for use on the Pacific Biosciences’ (PacBio) RSII, then ported and modified for operation on the new PacBio Sequel instrumentation platform.

Results: Characterization of the species-specific global composition of the tick microbiomes illustrates a complex microflora with high taxonomic diversity that varies depending on species and geographic locale within throughout California. Over 700 different microbial species were identified for each of the four tick species analyzed. Among these species were known mammalian pathogens, including Rickettsia monocensis, Franciella persica, Coxiella burnetii, and Borrelia burgdorferi. In addition, bacteria previously unassociated with ticks such as Micrococcus leutes, Williamsia serindens, Streptococcus suis, and Curtobacterium flaccumfaciens were identified.

Conclusion: Species-level taxonomic classification of tick microbiomes provides the granularity required to reveal a wide microbial diversity within tick species. Both expected TBD pathogens and novel microorganisms were identified.

Antibiotic Treatment Response Variation in Persistent Lyme Disease: Why Some Patients Improve while Others Do Not

Lorraine B Johnson, JD, MBA

There is considerable uncertainty regarding treatment of Lyme disease patients who do not respond fully to initial short-term antibiotic therapy. Choosing the best treatment approach and treatment duration for these patients remains challenging because treatment response among these patients varies, with some patients responding to treatment, while others do not. A previous study examined treatment response variation in a sample of over 3,500 patients enrolled in the MyLymeData patient registry, which was developed by That study used a validated Global Rating of Change Scale to identify three subgroups of treatment responders: non-responders, low responders, and high responders. The present study seeks to identify features that predict treatment response among these three groups using machine learning techniques. The majority of the features we identify relate to treatment approach. We then applied traditional statistical techniques to determine how these features relate to treatment response of non-responders, low responders, high responders, and patients who have recovered. High treatment response was most closely associated with the use of antibiotics or a combination of antibiotics and alternative treatments, longer duration of treatment, and oversight by a clinician whose practice focused on the treatment of tick-borne diseases.

It Ain’t Just One Thing

David Kaufman, MD; Ilene Ruhoy, MD, PhD

Chronic Lyme Disease (CLD) is a complex chronic illness. Controversy exists regarding whether it represents persistent Lyme infection or a post-infectious, possibly autoimmune syndrome, or a combination of both. This is an important topic as a greater understanding of CLD can help guide treatment options for these patients who suffer sometimes for decades and are often turned away from healthcare providers. Effective treatment has been notoriously difficult. Importantly, patients with CLD generally meet all the criteria for a diagnosis of ME/CFS. Interestingly as discussed below, these same patients very often present with similar signs, symptoms, and diagnoses that are seen in a large majority of ME/CFS patients regardless of any history of CLD. We will discuss the diagnostic concept of a Septad which includes Autoimmune disease, Mast Cell Activation Syndrome, Dysautonomia including small and large fiber neuropathy, Dysmotility/Dysbiosis/SIBO, hypermobility Ehler Danlos Syndrome (hEDS), Cranial Cervical Instability (CCI)/Tethered Cord (TC), and Infection including especially tick borne diseases, viral reactivation, and mycoplasma. The Septad concept provides a guide for both physician and patient regarding both the work up and the treatment plans. The identification of these particular entities can be made with objective data and can assist physicians in implementing management options. This presentation will briefly discuss each of these disorders including symptoms, evaluation, and possible treatment suggestions.

PANS & PANDAS: Novel Immunotherapy Transforming a Devastating Illness into a Treatable Disorder

Nancy O'Hara, MD

POTS, PANS & PANDAS are complex autoimmune diseases that require a multi-system approach. PANDAS (Pediatric Autoimmune Neuropsychiatric Disorder Associated with Strep) involves antibodies from a strep infection reacting with brain tissue (specifically the basal ganglia in the brain) and triggering an abrupt onset of immune dysregulation and movement, learning and behavioral problems including OCD, anxiety and tics. With PANS (Pediatric Acute-Onset Neuropsychiatric Syndrome), other infectious etiologies besides strep (yeast, viruses, other bacteria, parasites), toxic exposures, and metabolic abnormalities are among other potential triggers for the immune dysregulation resulting in an abnormal autoimmune reaction and negative behavior, physical, and cognitive changes. POTS or Dysautonomia can often be associated with these autoimmune disorders and also needs to be assessed and treated appropriately. Dr. O’Hara will discuss the latest research in the assessment and treatment of these devastating but recoverable illnesses. Participants will learn the research-based functional medicine approach to the care, diagnosis and treatment of these children.

Are Racial Disparities Seen in COVID also Seen in Lyme Disease?

Daniel Cameron, MD, MPH

Background: The numbers of Black people sick from COVID-19 have been greater than expected. Paradoxically, the numbers of Black people sick from Lyme disease have been less than expected. Black Lives Matter movement, an economic recession, and the COVID-19 pandemic have highlighted the impact of racial disparity.

Method: The factors responsible for these racial disparities will be examined.

Results: The causes for the racial disparity seen in the black community during this COVID-19 pandemic that have been proposed have included poor access to health care, the greater numbers work in essential services, delays in seeking medical advice, less access to personal protection equipment, and the financial costs of not working during a shelter in place order. The causes for the racial disparity in the Lyme disease community that have been proposed have included difficulty in recognizing the rash, lack of awareness of the significance of the rash, and poor access to care. Other causes for the racial disparity in the Lyme disease community will be discussed.

Discussion. Racial disparity is seen in COVID-19 and Lyme disease. The factors responsible for this racial disparity need to be identified. The racial disparities seen in COVID-19 and Lyme disease need to be addressed. We need a conversation on racial disparity for both COVID-19 and Lyme disease.

A Clinical Diagnostic System for Late-Stage Neuropsychiatric Lyme Borreliosis Based upon an Analysis of 100 Patients

Robert C. Bransfield, MD

Many late-stage chronic Lyme disease clinical findings are neuropsychiatric. A total clinical assessment is critical in diagnosis, especially since controversy surrounds the reliability of laboratory testing. The clinical findings of one hundred Lyme disease patients with chronic neuropsychiatric symptoms were entered into a database. The prevalence of each clinical finding pre-infection and post-infection was compared and calculated within the 95% confidence interval. Patients had minimal symptoms pre-infection, but a high post-infection prevalence of a broad spectrum of acquired multisystem symptoms. These findings included impairments of attention span, memory, processing, executive functioning, emotional functioning, behavior, psychiatric syndromes, vegetative functioning, neurological, musculoskeletal, cardiovascular, upper respiratory, dental, pulmonary, gastrointestinal, genitourinary, and other symptoms. The most prevalent symptoms included sustained attention impairments, brain fog, unfocused concentration, joint symptoms, distraction by frustration, depression, working memory impairments, decreased school/job performance, recent memory impairments, difficulty prioritizing multiple tasks, fatigue, non-restorative sleep, multitasking difficulties, sudden mood swings, hypersomnia, mental apathy, decreased social functioning, insomnia, tingling, word finding difficulties, name retrieval, headaches, sound hypersensitivity, paresis, anhedonia, depersonalization, cold intolerance, body temperature fluctuations, light sensitivity and dysfluent speech. The average patient had five symptoms pre-infection and 82 post-infection. Pattern recognition is critical in making a diagnosis. This study was used to develop three clinical assessment forms.

Lyme Disease, Substance Abuse and Addiction

Robert C. Bransfield, MD

Although a few journal articles have partially addressed the subject, the association between Lyme disease and substance abuse has never been thoroughly studied. It is an area that deserves attention. Many patients are inadequately diagnosed and treated which can contribute to patient feeling desperate for relief from multiple late stage symptoms. These symptoms can include insomnia, anxiety, depression, anhedonia, social anxiety, fatigue and chronic pain (headaches, musculoskeletal, neurological and autonomic). Late stage lyme disease patients are commonly treated with benzodiazepines for insomnia and anxiety and opioids for pain and some turn to alcohol, marijuana and other substances with addictive potential. As tolerance to these medications may occur. Disability, social isolation, and decreased purpose and meaning can further increase the risk for substance abuse. These contributors may lead to drug seeking behavior. It is possible that many overdose deaths may be attributed to inadequately diagnosed and treated Lyme disease. A recent study demonstrated only 1% (CI 0%-3%) of Lyme disease patients had substance abuse before acquiring Lyme disease while 12% (CI 6-18%) had substance abuse after acquiring Lyme disease. In addition, 41% of patients became very sensitive to the effects of alcohol after acquiring Lyme disease. It is important to address substance abuse when evaluating a patient with Lyme disease and to address this in the overall treatment planning. Interestingly, the same medications used to treat addictive disorders are also used to treat symptoms associated with late stage Lyme disease. These medications include disulfiram, naltrexone, topiramate, clonidine, gabapentin, acamprosate, baclofen and bupropion.

Neuropsychological Interventions of Tick-Borne Diseases

Leo J. Shea III, PhD

Discussion will center on an understanding of how neuropsychological evaluation is critical to an understanding of the cognitive emotional and behavioral components of those patients diagnosed with tick-borne diseases.

Medication Strategies for Neuropsychiatric Lyme Disease

Linda Williams, MD

The Role Inflammation and Glutamate Play in Psychiatric complaints and How to Utilize Medications and to Promote Healing and Alleviate Suffering.

Increased Inflammation has been implicated in a number of neuropsychiatric illnesses. In addition, there is evidence that altered glutamate transmission effect patients in a number of ways including pain, sleep, mood disorders, anxiety and seizures. In this talk we will look at the way that infection plays a role in inflammation as well as in alterations glutamate transmission There is evidence that inflammation can cause glutamate excess in the nerve synapse and can increase levels of quinolinic acid. We will examine the disorders related to this and treatment strategies for both activating disorders such as anxiety and seizures as well as sleep, and cognitive complaints. We will review using medications not just as they are labeled by the FDA, but thinking about them according to their mechanism of action and utilizing them as tools to help approach the healing of main psychiatric complaints that present in Lyme disease patients.

Preventing Lyme and Other Tick-Borne Diseases

Alexis Chesney, ND

As providers are challenged by growing numbers of complex Lyme and Tick-Borne Disease cases and in recognition of the interconnections between humans, animals, plants and their shared environment, tick bite prevention is imperative. In this presentation, I will review the tick species of North America, how to identify them and what pathogens they may carry. Tick attachment times and pathogen transmission have been documented. Tick habitat and tick control interventions will be discussed. Recognizing how Cryptolepis has been used as an antimalarial prophylaxis can inform anti-tick-borne disease prophylaxis.

Babesiosis and Theileriosis – Update on Current Diagnostic Methods

Jyotsna Shah, PhD

Piroplasmosis is a tick-borne disease that is caused by infection with apicomplexan protozoa, such as the Babesia or the Theileria species. These protozoan parasites are transmitted by vector ticks and can infect many different species of domestic and wild animals. Some species of the Babesia protozoan parasites are also pathogenic to humans. In the US, B. microti, and B. duncani (formally WA-1) are the most common pathogenetic species of piroplasms causing human infections. Recent reports suggest that some Theileria species may also cause severe acute diseases in humans as human theileriosis. Due to the wide spectrum of Babesia and Theileria species and their species-specific characteristics with relation to disease severity, transmission, epidemiology, and drug susceptibility it is important to accurately identify the causative agents. Thus, the main methods currently used for the detection of Babesia and Theileria spp. for diagnostic purposes, including Fluorescent In Situ Hybridization (FISH) will be discussed.

Innovative Bartonella Treatments - Clinical Experience Applying Laboratory Research

Thomas A Moorcroft, DO

Discuss my clinical experience with utilizing emerging in vitro Bartonella stationary form treatments in clinical practice, including the use of oral, IV and iontophoresis patches in adults and children. The ability to utilize topical application of treatments, such as methylene blue, is a promising clinical innovation to help younger children who are unable to swallow capsules be able to receive much needed treatment Bartonella stationary forms. This will include discussion of clinical experience with IV methylene blue and glycyrrhizzic acid.

Lyme & TBD’s in Pregnancy: Risks, Treatment Options

Richard I Horowitz, MD

Babesia is a single celled protozoan parasite which may be transmitted through the bite of an infected tick, blood transfusion and/
or maternal-fetal transmission. We describe the case of a woman previously treated for Lyme disease and babesiosis who relapsed
with severe malaria-like symptoms during the 3rd trimester of two consecutive pregnancies. Testing for active babesiosis was
positive in both pregnancies despite prior conventional therapies, and she was subsequently treated with high dose atovaquone
(1500 mg PO BID) and Zithromax during each of her 3rd trimesters, along with clindamycin during her 3rd pregnancy. At term,
neonates were healthy with no clinical evidence of active babesiosis (no fever, acute respiratory distress syndrome [ARDS],
hepatosplenomegaly or jaundice) and no laboratory evidence of babesiosis (no positive blood smears, hemolytic anemia,
neutropenia or thrombocytopenia). Intramuscular benzathine penicillin was simultaneously used throughout her four pregnancies
to prevent transmission of Borrelia burgdorferi, the agent of Lyme disease, with no evidence of transmission of the spirochete
to the fetuses. Although this novel Lyme and Babesia therapy was effective in preventing congenital transmission of Borrelia
burgdorferi and Babesia microti in consecutive pregnancies, future studies are needed to define the optimal management strategy
for pregnant patients with Lyme disease and babesiosis due to the possible relapsing, remitting nature of the disease and the
potential for associated morbidity and mortality.

Association between Tick-Borne Illness and Neurodegeneration - A Systematic Review

Omar Morales, MD

ALS, MS, Other CNS diseases associated with Lyme: Anatomopathological alterations in neurological complications of Lyme disease; charts of neurological Lyme disease signs and symptoms; neuropathies with abnormal myelination in Chronic Lyme borreliosis and differential diagnosis.

Aim: The goal of this lecture is to educate practitioners about the neurological involvement in Lyme disease, showcase anatomopathological changes of neural tissues, provide signs and symptoms of neurodegeneration in chronic Lyme disease. It will also explain the demyelinating variables observed in Lyme borreliosis and possible course of action for both general physicians and specialists. Environmental and predisposing factors: How infections, environmental toxins, inflammation, formation of free radicals can contribute to myelin sheath damage.

Neurodegeneration Process: Explanation of the neuro-immunological degeneration process as a consequence of a triggered system.

The Role of Genetics: Overview of the genetic variations associated with cellular stress.

ALS, MS, Other CNS diseases associated with Lyme: History, research and conceptualization of neurodegeneration, demyelination, neuropathy & denervation.

Autoimmune Neurodegeneration: Overview of underlying trigger factors and immune ablation.

Additional Factors to Consider: Lyme life philosophy and basic recap of treatment pillars for the chronically ill.

The Blood Brain Barrier (BBB) and Neuroinflammatory Disorders

Kenneth A Bock, MD

The blood brain barrier (BBB) is a key regulatory interface between the CNS and peripheral tissues, especially the immune system, and its integrity helps maintain proper function of the CNS cells it protects and nourishes. It does this via the neurovascular unit (NVU), which is a complex functional and anatomical structure composed of brain endothelial cells and their tight junctions, basement membrane with overlying pericytes, astrocyte end-feet, and neurons. It is affected by cells and substances from both the peripheral circulation, as well as the CNS side. Disruption of the BBB is seen in neurologic disorders such as ischemic stroke, traumatic brain injury, and Alzheimer’s disease, neuroinflammatory disorders such as MS, and neuropsychiatric disorders such as bipolar disorder. Increased permeability of the BBB allows entrance of inflammatory cells and substances into the CNS, with subsequent neuroinflammation and clinical sequelae. In turn, this BBB disruption and dysfunction can be related to inflammatory mediators that enter the circulation due to altered intestinal permeability.

In adolescents, neuroinflammatory disorders are becoming increasingly recognized as a basis for neuropsychiatric disorders, with symptoms that include tic disorders, OCD, anxiety, and mood dysregulation, among others. This disorder has been given numerous names, including, but not limited to PANDAS (Pediatric Autoimmune Neuropsychiatric Disorder Associated with Strep), PANS (Pediatric Acute Onset Neuropsychiatric Syndrome), and ITAE (Infection Triggered Autoimmune Encephalitis). This talk will address the importance of the blood brain barrier in maintaining homeostasis and preventing neuroinflammation, as well as discuss the importance of the gut/brain axis, with a specific focus on these alterations in adolescents.

Synergistic Treatment of COVID-19 with Stem Cells and Peptides

Kent A. Holtorf, MD

The end of 2019 marked the beginning of a new era and challenge for us here in the US and across the world when the initial SARS-COV-2 infection was reported in the city of Wuhan, Hubei province, China. The highly contagious positive-stranded RNA virus has rapidly spread to become a global health crisis on a pandemic scale. The virus mainly targets pulmonary epithelial cells via a spike protein that binds to the host’s ACE2 receptors.

While many younger healthy patients have little or no symptoms, aged individuals and those with significant comorbid conditions are particularly susceptible to severe disease and high mortality risk. The virus causes an exaggerated immune response that results in a vicious cycle of proinflammatory cytokine production, severe lung, and multi-tissue damage with resultant ARDS with an inability to oxygenate tissues of the body.

Despite the high incidence of mortality with ARDS, no specific effective treatment has emerged in 40 years.1 Stem cells exhibit anti-inflammatory, immunomodulatory, and reparative effects, as well as having significant antimicrobial properties. As would be expected, stem cell therapy has shown promise in the ability to increase survival, reverse the exaggerated immune response, heal injured lung tissue via a paracrine action and reduce overall inflammation, changing the immune system from a Th17/Th2 to a Th1/Treg dominant pattern.

Following some encouraging case reports2 and small studies that showed rapid, substantial improvement with critically ill COVID-19 patients, mesenchymal stem cells (MSC’s) have emerged as a promising tool for moderate to severe COVID-19 patients.3

Peptide therapy is also an emerging therapy that holds significant promise with both chronic illness and severe acute illness. While many peptides could be discussed, this presentation will concentrate on two classes of peptides, the thymic peptides, which include thymosin alpha 1, thymosin beta 4, and thymosin and the antimicrobial peptide LL-37.

The thymosins are shown to also have significant immune-modulatory, anti-inflammatory, regenerative, and antimicrobial effects, so they would seem to be an ideal addition to stem cells. They work very synergistically together. The peptides stimulate and activate the stem cells, and the stem cells, in turn, secrete immune-modulatory and antimicrobial peptides.

This lecture will review the current literature on the use of these emerging therapies for chronic and acute infections, ARDS, and specifically in the treatment of COVID-19.

Preventing the Failed Patient

Eric Gordon, MD and Nafysa Parpia, ND

Patients with Chronic Tick-Borne Disease who do not respond in an expected manner to therapy are almost always those who have a group of associated disorders. These include a tendency towards Mast Cell Activation which is often confused with a Herx, an increased environmental toxicant load and/or most commonly a decreased ability to metabolize and safely excrete exogenous and endogenous toxins.

These processes either all lead to and/or are initiated by chronic dysregulation of the immune system, which in turn may lead to neurologic symptoms. When we layer the combination of detoxification, antibiotics and immune modulators we achieve good patient outcomes in people who we were unable to help when these therapies were not combined.

COVID-19: Prevention, Diagnosis, Treatment Options

Richard I Horowitz, MD

Asymptomatic or minimally symptomatic infection with COVID-19 can result in silent transmission to large numbers of individuals, resulting in expansion of the pandemic with a global increase in morbidity and mortality. New ways of screening the general population for COVID-19 are urgently needed along with novel effective prevention and treatment strategies.

A hypothetical three-part prevention, diagnostic, and treatment approach based on an up-to-date scientific literature review for COVID-19 is proposed. Regarding diagnosis, a validated screening questionnaire and digital app for COVID-19 could help identify individuals who are at risk of transmitting the disease, as well as those at highest risk for poor clinical outcomes. Global implementation and online tracking of vital signs and scored questionnaires that are statistically validated would help health authorities properly allocate essential health care resources to test and isolate those at highest risk for transmission and poor outcomes.

Second, regarding prevention, no validated protocols except for physical distancing, hand washing, and isolation exist, and recently ivermectin has been published to have anti-viral properties against COVID-19. A randomized trial of ivermectin, and/or nutraceuticals that have been published to support immune function including glutathione, vitamin C, zinc, and immunomodulatory supplements (3,6 Beta glucan) could be beneficial in preventing transmission or lessening symptomatology but requires statistical validation.

Third, concerning treatment, COVID-19 induced inflammation and “cytokine storm syndrome” with hemophagocytic lymphohistiocytosis (HLH)/Macrophage Activation Syndrome (MAS) have resulted in extreme morbidity and mortality in those with certain comorbidities, secondary to “acute respiratory distress syndrome” (ARDS) and multiorgan dysfunction with disseminated intravascular coagulation (DIC). Deficiency in red blood cell, serum and alveolar glutathione has been published in the medical literature for ARDS, as well as viral and bacterial pneumonias, resulting from increased levels of free radical/oxidative stress. A randomized controlled trial of blocking NF-κB and cytokine formation using glutathione precursors (N-acetyl-cysteine [NAC] and alpha lipoic acid) and PO/IV glutathione with associated anti-viral effects should be performed, along with an evaluation of Nrf2 activators (curcumin, sulforaphane glucosinolate) which have been scientifically proven to lower inflammation. Since high mortality rates from sepsis induced DIC due to COVID-19 infection has also been associated with thrombotic events and elevated levels of D-dimer, randomized controlled trials of using anticoagulant therapy with heparin is urgently required. This is especially important in patients on ventilators who have met certain sepsis induced coagulopathy (SIC) criteria. The use of acetazolamide with or without sildenafil also needs to be explored with or without heparin, since increased oxygen delivery to vital organs through prevention of thrombosis/pulmonary emboli along with carbonic anhydrase inhibition may help increase oxygenation and prevent adverse clinical outcomes.

Conclusion and Implications
A three-part prevention, diagnostic, and treatment plan is proposed for addressing the severe complications of COVID-19. Digital monitoring of symptoms to clinically diagnose early exposure and response to treatment; prevention with ivermectin as well as nutritional therapies that support a healthy immune response; treatment with anti-inflammatory therapies that block NF-κB and activate Nrf2 pathways, as well as novel therapies that address COVID-19 pneumonia and ARDS with DIC including anticoagulation and/or novel respiratory therapies with or without acetazolamide and sildenafil. These three broad-based interventions urgently need to be subjected to randomized, controlled trials.

Mast Cell Targeted Treatment in Patients with Tick-Borne infections, MCAS and COVID-19

Tania Dempsey, MD

Background: While the full spectrum of disease resulting from COVID-19 is not known, it is becoming clearer that some patients suffer worse sequelae than others. In addition, there is emerging evidence for the development of post-viral or post-COVID syndrome in a subset of patients. It is known that infections, including viral ones, can trigger mast cell activation and subsequent mediator release that can lead to localized as well as systemic manifestations. Whether Mast cell activation syndrome is partly or fully responsible for the sequelae of COVID or post-COVID syndrome is unknown but needs to be understood.

Aims: Goal
To understand whether patients with mast cell activation syndrome, triggered or worsened by chronic/persistent Lyme and/or tick-borne infections, fare better or worse from COVID or develop post-COVID syndrome.

Patients and Methods: 5 patients with a history of tick-borne infections and mast cell activation syndrome, , were diagnosed with COVID-19 between March 16 through May 1, 2020. 3 out of 5 patients were confirmed by PCR while the other 2 had clinical symptoms consistent with the infection. One of the two with negative PCR has been confirmed to have SARS-CoV2 IgG antibodies. Four of the patients had well controlled MCAS prior to the infection and were not on any treatment for their past tick-borne infections. One patient was being actively treated for Babesiosis and Lyme disease with Disulfiram and was on MCAS medications but was not well controlled.

Results: All patients received some form of treatment for COVID-19. 2 were treated with hydroxychloroquine with azithromycin, 2 were treated with ivermectin, 1 only received supplements for immune support. The four patients that were well controlled on adequate mast cell medications, including H1/ H2 blockers and additional agents, depending on the patient. They had considerable variability in their clinical course, but they had less respiratory symptoms during the illness and none have evidence of residual symptoms. One patient on Disulfiram, had a more symptomatic course, including severe shortness of breath and cough, but was able to avoid hospitalization. She was treated with Hydroxychloroquine and azithromycin with worsening symptoms after the medications were discontinued. Her clinical course improved with the addition of oral Benadryl, nebulized b-agonist, montelukast and restarting of hydroxychloroquine. All patients have recovered without evidence of long-term sequelae.

Conclusion: Treatment targeted to mast cell blockade and/or stabilization may decrease severity of COVID19 in patients with a history of Lyme disease and other tick-borne infections.

The Importance of Communication between Physicians and Society in Prevention of Lyme Disease

Nataliya Banadyha, DSc, PhD, MD

Background: The seasonality of manifestations is important among infectious pathology, as well as its targeted prevention. At the same time, it is important to disseminate medical information about the prevention of certain diseases among population. In recent years, sanitary and educational work aimed at preventing tick-borne diseases has developed in Ukraine.

Lyme disease (LD) in children has difficulties in terms of prevention, diagnosis and treatment. The essence of the problem is marked by the climatic and geographical features of the city of Ternopil, the lack of practice of systematic processing of park and forest strip from ticks. The medical aspects of this pathology are primarily determined by the lack of common approaches to diagnosis and treatment in European countries [2,6] and, for example, in the United States[1,3-5]. However, all researchers focus primarily on preventative measures and adequate antibacterial therapy.

Aims: Frequency analysis of tick bite treatment and confirmation of LD among Ternopil children for the period 2015-2019 were performed.

Patients and Methods: Medical and disease histories were collected in detail, clinical examination of the patient and laboratory diagnostics were carried out: PCR of ticks to identify the pathogen; ELISA of blood (Ig M and Ig G to Borrelia burgdorferi). A recommendation notes on how to deal with tick bites was distributed to the general public and health care professionals in 2017. Detailing of the actions of parents and the physician's diagnostic and treatment tactics, specific trainings and seminars were conducted.

Results: It was found that during the observation of the tick bites, 271 children went to the doctor, then in 75 (27.67%) were diagnosed the LD. Due to the educational events that took place in 2017, we analyzed the frequency of visiting a doctor. We found out that, following the introduction of a unified approach to: tick removal, primary prevention; the incidence of LD has decreased significantly. Namely, in 2015 confirmed LD was in 19 children, in 2016 – 10, 2017 – 23, 2018 – 12, 2019 – 10 patients. At the same time, the frequency of calls to a doctor increased significantly: 2015 – 47 children, 2016 – 46; 2017 – 56; 2018 – 62; 2019 – 87 people.

Conclusions: Educational work improves timeliness of diagnostics, determines rational antibacterial prophylaxis, reduces morbidity. It motivates to further improve the forms and means of educational activities among the general public and medical staff to improve the situation.

Co-Infections Patterns in Post-Treatment Lyme Disease Patients

Gisell Brenton, MD

Background: Up to 60% of patients with Lyme disease (LD) present more than one bacterial or viral co-infection. Currently, the only approved treatment for tick-borne diseases (TBD) is several antibiotics courses. Serology testing is controversial for a follow-up (FU) assessment in patients with TBD.

Aims: A new comprehensive treatment program for LD patients has been developed, and it is based on multidisciplinary efforts and personalized treatment to promote immunomodulation, and therefore enhancement of tick-borne pathogens recognition and eradication. This research protocol report aims to compare tick-borne associated pathogens in patients with LD, before and after enrolling in the Lyme Disease and Chronic Infections Program (LDCIP) at Sanoviv Medical Institute.

Patients and Methods: The LDCIP at Sanoviv starts with promotion of oxidative stress with hyperbaric therapy, ozone autohemotherapy, UV therapy, artesunate, and high doses of intravenous vitamin C. The program continues with whole-body hyperthermia (WBH) along with intravenous antibiotics. After WBH, patients receive hydration therapy and antioxidation support, finally end with detoxification. We analyzed patients enrolled in the LDCIP in 2019 with pre and post-treatment laboratory tests for co-infections of LD (n=14). The tick-borne associated pathogens analyzed were Babesia microti (Bm), Bartonella henselae (Bh), Ehrlichia chaffeensis (Ec), Mycoplasma pneumonia (Mp), and Rickketsia akari (Ra); we also described viral infections which included Epstein Barr virus (EBV), Coxsachie virus (CV), and Human parvovirus B19 (HPB19).

Results: 64% (n=9) were male patients and the mean age of presentation was 40 years (range 20-58 years). Nine patients (64%) did not present any co-infection and five patients (36%) presented at least one co-infection before treatment. Eight weeks after treatment, we observed seven patients (50%) that remain with negative results for co-infections, and seven patients (50%) were positive for at least one co-infection. All patients with pre-treatment bacterial and protozoa co-infections decreased except for Bh, which also resulted present in a patient previously negative. All patients with viral infections before treatment presented negative post-treatment results, except for EBV, which remain in patients previously infected and also was the most frequent persistent infection in FU assessment.

Discussion: This study has provided evidence that different serology patterns exist in LD patients after receiving comprehensive treatment. EBV infection should be screened in LD patients with relapsing symptoms. Further studies are needed to confirm this FU pattern in LD patients.

At-Home Mold Testing vs. Professional Mold Testing: Setting the Plan for Immunocompromised Individuals

Corey Levy, CMI, CIE

In the wake of COVID-19, keeping our immune systems strong has never been so important. This has been a constant struggle for those who are immunocompromised, and now they are faced with a new challenge. As we have all been needing to spend more time inside our homes many have begun to notice their environment may be impacting their health. With the growing number of individuals who are immunocompromised, the demand for testing their homes for certain indoor pathogens is at an all-time high. This presentation is aimed to help medical practitioners understand how to advise their patients on the best at-home sampling methods and when an Indoor Environmental Professional should be brought in to perform an inspection.

Lyme Disease and Associated Immune Dysfunction

Sagar Matharu, BS

Background: Past studies have ambiguously linked Lyme Disease to weakened immunity. However, with the outbreak of the novel coronavirus and it’s own implications, understanding the immunological consequences of Lyme Disease offers insight into the viral immunity and susceptibility of comorbidity and its corresponding pathology.

Aims: The purpose of this study was to draw lines and establish the relationship between Lyme Disease and a patient’s viral immunity.

Patients and Methods: Retrospective studies were conducted on 311 patients with confirmed Lyme Disease from January 2019 to June 2020. Data was taken from patients from the Lyme endemic region of Maryland and analyzed at multiple laboratories for clinical use. P-values were obtained by comparing CD4/CD8 ratios, absolute CD8 counts, EBV and HHV6 viral titers, and immunoglobulin assays.

Results: TIn a cohort of 311 patients, 241 patients had positive IgG 41 bands and an associated mean CD4/CD8 ratio > 2.50. 250 patients had elevated EBV titers and 246 had elevated HHV6 titers. In comparing the positive and negative t-tests, the p value on both a one tailed and two tailed test was shown to be p<0.0001 across all analyses.

Discussion: Immune-deficiency, defined as having a compromised immune system, was significant in patients with Lyme Disease. Clinically, lyme patients returned elevated CD4/CD8 ratios during activation of an immunoglobulin G 41 band. Those same patients subsequently had elevated EBV and HHV6 viral titers. Not all patients showed glaring immunological abnormalities, however, the statistical significance implicates Lyme disease as a factor of immune deficiency. Additionally, the immuno-compromise may leave patients more susceptible to COVID-19.

Doing Disulfiram; Experience from 9/1/2019-6/12/2020

Hope McIntyre, MD

BACKGROUND: Based on Dr Kenneth Leigner’s published 3 patient case study and anecdotal reports of effectiveness from many other patients and health care providers, I began using disulfiram for the most difficult to treat patients in my own practice in September of 2019. The treatment seemed to be working, but some patients were experiencing side effects. Also the treatment seemed to work best for patients with more conclusive evidence for Lyme disease.

AIMS: I set out to examine the effectiveness and side effects of disulfiram in my own practice.

METHODS: Using my electronic health record as well as a list from our compounding pharmacies, 113 patients were identified for whom I had prescribed disulfiram. Patients were included in the analysis if I had diagnosed them with either Lyme disease or babesiosis, and if they had taken the medication at an adequate dose for longer than a month. Most patients were still on treatment at the end of the analysis. The resultant study groups were 9 children age 12-17 years at the time of treatment and 48 adults 18 years and older. Patients were asked to rate their level of wellness from 0-10 at each visit and their pain from 0-10 at each visit. Based on my chart evaluations 4 result groups were identified: not improved, slightly improved, improved, and dramatically improved. A CBC with diff was performed on patients every 3-4 weeks.

RESULTS: Of the 9 children 6/9 (78%) were dramatically improved, 1 slightly improved(11 %) and 1 not improved(11 %.) in a subgroup of children who met a strict definition for Lyme disease(they had either a physician diagnosed Lyme disease rash with at least a positive titer and IgM WB through a standard laboratory, OR a positive titer with a positive IgG WB through a standard laboratory) the numbers were slightly better. 4/5 (80%) dramatically improved, and 1/5 (20 %) slightly improved. None of the 9 children had significant laboratory abnormalities requiring ending treatment. Of the 48 adults, 9/48 (19 %) were dramatically improved, 23/48(48 %) were improved, 11/48(13 %) were slightly improved, and 5/48(10 %) were not improved. When the above more strict criteria for Lyme were applied a subgroup of 11 patients was identified of which 3/11(27 %) were dramatically improved, 4/7(36 %) were improved, 2/11 (18 %) were slightly improved an 2/11(18 %) were not improved. 2 4(8 %) adults had to stop treatment or decrease the dose due to elevated liver function tests. All of these resolved off the treatment. 1(2 %) of adults had to stop treatment due to overwhelming fatigue. 3(6%) of the adults had to stop the medication or decrease the dose due to pressured speech and racing thoughts consistent with mild mania. 2(4 %) stopped due to numbness and tingling. 1(2 %) stopped the treatment due to sudden severe unexplained anxiety

DISCUSSION: Disulfiram yielded improvements in both children and adults which were more than the 30 % expected with placebo. Adults seemed to have more side effects than children. The drug did seem to work better in those with a more strict definition of Lyme disease, especially in the children. Based on this evaluation, I will continue to use disulfiram in my most difficult to treat patients while being careful to monitor for side effects and laboratory abnormalities. Note that these were my “most difficult to treat patients” who had either not responded to more usual treatment or were relapsing off other treatments, so that these results may be actually lower than would be found in a ‘less difficult to treat” cohort.

Development and Validation of a Droplet Digital PCR Assay for the Detection and Quantification of Bartonella Species within Human Clinical Samples

Jennifer Miller, PhD

Development and validation of a droplet digital PCR assay for the detection and quantification of Bartonella species within human clinical samples

Background: In recent years, the development of droplet digital PCR (ddPCR), a new molecular technology, has enabled an unprecedented expansion of DNA amplification capabilities for research and diagnostic applications, not just in the area of cancer and gene expression, but also most recently in areas of infectious disease diagnosis.

Aims: Develop, optimize, and validate a ddPCR assay, using a QX200 Droplet Digital PCR (ddPCR™, Bio-Rad,Hercules, CA) system, for the detection of Bartonella spp. DNA within several sample matrices, including in vitro infected epithelial cell cultures and pre-characterized clinical blood samples from patients with or without documented Bartonella spp. bacteremia.

Methods: The Bartonella spp. ddPCR assay was developed based upon previously published TaqMan-based qPCR assays that can amplify DNA of over 25 Bartonella spp. Host DNA (housekeeping gene) amplification serves as a reference target to facilitate quantification. The efficiency, sensitivity, and specificity of the Bartonella spp. ddPCR assay was assessed by direct comparison with the current qPCR method used by the Intracellular Pathogens Research Laboratory (North Carolina State University, North Carolina, USA), and Galaxy Diagnostics (Research Triangle Park, North Carolina, USA). Three matrices were tested to optimize the Bartonella spp. ddPCR assay: DNA from human blood samples spiked with Bartonella DNA, experimentally infected human cell lines, and pre-characterized clinical human blood samples. DNA extraction was performed using either the Qiagen DNeasy Kit (tissue DNA extraction) or QIAsymphony DNA Kit.

Results: Bartonella spp. ddPCR assay parameters were successfully optimized to detect Bartonella concentrations equivalent to 0.5 bacterial genome copies per microliter of blood (0.001 pg/ul of bacterial DNA). The number of droplets detected (resolution) for each concentration was consistent across each of four assessed time points. The Bartonella spp. ddPCR assay detected 16 species/strains for which isolates or DNA was available for testing: B. henselae; B. quintana; B. vinsonii subsp. berkhoffii (genotypes I, II, III and IV); B. vinsonii subsp. vinsonii; B. melophagi; B. volans; B. monaki; B. alsatica; B. bovis; B. elizabethae; B. clarridgeiae; and B. koehlerae. Bartonella DNA was detected in only one previously negative patient sample (119/120 negative; 99% specificity). ddPCR sensitivity (53/112) was substantially better than qPCR (6/112) when testing patient blood and enrichment blood culture samples.

Discussion: The assay described in this abstract is the first step toward the development of a multiplex ddPCR assay (i.e. using the QX One from Bio-Rad) for the simultaneous detection and absolute quantification of multiple vector-borne pathogens (such as Babesia, Bartonella and Borrelia) within clinical samples.

The Potential Role of NADPH Oxidase and Heme Oxygenase 1 in COVID-19 Associated Cytokine Storm

Robert Miller, ND

Bile has been rapidly gaining interest as an indispensable facet of detoxification, potentially contributing to the elimination of Lyme-associated pathogens. Previous analyses have found an increased frequency of single nucleotide polymorphisms (SNPs), related to functional and detoxification pathways, in individuals with chronic Lyme disease.

The current analysis was performed to evaluate genetic variants involved in mast cell regulation, and bile synthesis and utilization in participants with chronic Lyme disease.

Patients and Methods:
To determine if unique genetic patterns exist within chronic Lyme patients, we analyzed mast cell- and bile-related genes from 470 participants, who submitted their 23andme genome for a global contrast to data supplied by the 1000 Genomes Project phase 3. Eligible participants had been diagnosed with chronic Lyme disease, and together encompassed a diverse demographic background. Reference and alternate alleles for each SNP was determined using the HaploReg v4.1 database. The ratio of SNPs involved in mast cell regulation, and bile synthesis and utilization was compared between the chronic Lyme group and the 1000 Genomes Project phase 3 data.

The analysis revealed an increased prevalence of minor allele variants in the NR1H4, and ABCB4 genes in the chronic Lyme disease group. Specifically, two SNPs in NR1H4 were found to be increased by 1.53 and 1.8 %, respectively: one SNP in ABCB4 was found to be increased by 1.51 %.

Discussion: T
To our knowledge, the present analysis is an unpreceded evaluation of bile-related genes in regard to chronic Lyme disease. Bile synthesis and utilization may be critical for the elimination and detoxification of Lyme-inducing pathogens, and associated inflammatory byproducts. NR1H4 encodes for the farnesoid X receptor, known as FXR: a bile acid-activated transcription factor critical for the regulation of bile synthesis, homeostasis, and enterohepatic flow. Multidrug resistance 3 (MDR3), encoded by ABCB4, is a floppase, which translocates phosphatidylcholine to the outer leaflet of the cellular membrane for bile synthesis, and minimizes bile salt toxicity. Mutations in ABCB4 have been previously associated with impaired bile flow, biliary lecithin output, micelle formation, and excess hydrophilic bile synthesis, resulting in membrane damage. The closely related protein MDR1 has been found to regulate the metabolism, and efficacy of various compounds. Further genetic and computational analysis is warranted to further elucidate a potential role of MDR3 and FXR in chronic Lyme disease.

Increased Mutations in Genes Related to Bile Utilization and Synthesis in those with Chronic Lyme Disease

Robert Miller, ND

Background: It has been generally accepted SARS-COV-2, the viral agent responsible for coronavirus disease 2019 (COVID-19), gains cellular entry via angiotensin converting enzyme 2 (ACE2), which degrades angiotensin I (Ang I) and angiotensin II (Ang II) to angiotensin 1-9, and 1-7, respectively. Furthermore, the currently accepted consensus is that SARS-COV-2 likely downregulates ACE2, via induced ectodomain-shedding, similar to the highly homologous SARS-COV.

Aims: To evaluate potential molecular pathways involved in COVID-19 pathology, the present analysis reviewed relevant literature related to hallmark indicators of disease severity. The selected clinical parameters were then considered in the context of known pathways related to the various COVID-19 features.

Methods: A literature review was conducted within the PubMed and Google Scholar databases, using several combinations of key terms: including, “COVID,” “COVID-19,” “SARS-COV,” “SARS-COV-2,” “severity,” “ACE2,” “Angiotensin II,” “NADPH oxidase,” “NOX,” “TLR,” “Nf-kB,” “Interlukin-6,” “IL-6,” “Ferritin,” “Bilirubin,” “Biliverdin,” “HMOX-1,” “Heme oxygenase,” “HO-1,” and “Hypokalemia.” Several clinical manifestations reported in COVID-19 patients were selected for consideration based on their relevance to disease severity, and outcomes.

Results: Several analyses have found various laboratory markers were significantly associated with fatality, mechanical ventilation, or respiratory failure: notably, interlukin-6 (IL-6), C-reactive protein (CRP), ferritin, and bilirubin. Multiple reports have emerged with similar findings, with a particular emphasis on IL-6 due to its’ critical role in pulmonary inflammation. Hypokalemia has also been reported in severe patients, supporting the notion of potential ACE2 downregulation. During our critical evaluation, the bidirectional Ang II-NOX, Ang II-Nf-kB, TLR-NOX, and Ang II-NOX-HMOX-1 pathways appeared to have exceptional potential to be involved in COVID-19 pathology.

Discussion: The potential role of NADPH oxidase (NOX), and heme oxygenase-1 (HO-1) in COVID-19 has not been previously evaluated to our knowledge. Angiotensin II, NOX, and IL-6 have shown to have bidirectional relationships within various tissue types. NOX synthesizes the potent oxidants superoxide, and hydrogen peroxide. SARS-COV-2 has been further reported to activate toll-like receptors (TLRs), which also stimulate NOX in numerous tissues. NOX2 inhibition has been repeatedly shown to reduce virus-induced inflammation, and viral titers in vivo. Heme oxygenase-1 (HO-1) metabolizes heme into iron, and biliverdin, which are subsequently converted to ferritin and bilirubin: carbon monoxide (CO) is released as a byproduct. However, bilirubin and CO have been shown to have to inhibit NADPH oxidase. Thus, it is hypothesized the overexpression of HO-1, an innate antiviral and cytoprotective mechanism, and NOX may contribute to COVID-19 pathology.

The Role of Anaerobic Bacterias as Intersection between Lyme Disease and COVID-19

Albin Obiltschnig, MD

Anaerobic bacterias, particularly Archaeas, appear to play a significant role as a portal of entry in COVID-19 infections.
Immediate initiaton of Anaerobex (Metronidazol) as a causal treatment at the onset of the first COVID-19 symptoms has had positive effects in high-risk patients.
All my patients had previously suffered from Lyme disease, so in the months prior to the emergence of the Coronavirus crisis, they had required several courses of long-term antibiotic treatment. This treatment, of course, allows preferential proliferation of anaerobic bacterias on the mucous membranes of the upper respiratory tract, leading to colonization, which is, in part, nonpathogenic. Colonization of the oral cavity, pharynx and bronchi with anaerobic bacterias, including Archaea, has increased dramatically.

A Serological Alternative: An ACWOP-Based Point of Care Sensor for Infectious Diseases with Potential Application in Lyme Disease Detection

Andy Sanchez, MS

According to the WHO, infectious diseases account for 3 of the top 10 causes of death for humans, a threat to global health made even more salient by the recent Coronavirus pandemic. Whether it’s the need to implement effective contact tracing in a pandemic or the need to more reliably screen and treat Lyme disease patients during the preliminary stages of infection, infectious disease monitoring is a critical tool in modern public health.

Traditionally, clinical detection of infectious diseases is performed using an ELISA, which captures specific antibodies from a subject’s serum on an antigen-coated surface. These antibodies are then read out using a tagged secondary, which binds to the subject’s primary antibodies in a species-dependent manner. This secondary incubation comes with time and resource costs, as well as limitations when tracking and detecting diseases which affect multiple species.

We have developed a novel sensor which bypasses these limitations and could enhance infectious disease monitoring. Our device incorporates the inherent catalytic activity of antibodies, which allows antibodies to convert water to hydrogen peroxide in a process known as the Antibody-Catalyzed Water Oxidation Pathway, or ACWOP. An ACWOP sensor allows for the direct detection of immobilized antibodies, eliminating the need for secondary antibodies and associated time and resource costs. Because ACWOP works for all antibodies, this sensor could be applied as a screen for many major diseases and could provide a consistent technical detection system across different species, allowing for an expedited transfer of information gathered in different contexts.

In this presentation, I will demonstrate an early stage ACWOP sensor which uses cheap, simple reagents, relevant for point-of-care applications. Our proof-of-concept experiments demonstrate a clinically-relevant limit of detection for our sensor. We have begun preliminary experiments which adapt this sensor for use in detecting commercially available C6 antibodies. We believe this work could pave the way for a major alternative in infectious disease monitoring across a host of animals and diseases.

Pregnancy and Lyme Disease: Analysis of Literature and Guidelines

Diana Uitdenbogerd-Drenth, PhD

Foundation Tick Bite Diseases NL commonly receives questions about the risks and what to do during pregnancy when mothers have Lyme Disease. We hear about children recently born with congenital Lyme Disease and from adults of all ages who state that they had the disease from their mother.

We investigated what the scientific literature actually claims, and which articles are important an which not and why the risk is surprisingly underestimated, as well as what women can do during pregnancy.

We searched Pubmed for Congenital Lyme Disease and made a shift in reviews, case studies, population studies and guidelines. We checked with at least 10 lists of patient-organizations if we had missed an article. We had access to most articles and read them in full.

The Dutch guideline states that there is a small chance of congenital LD, based on 5 case studies and 4 population studies (2 retrospective, 2 prospective). Included population studies however are often statistically not sufficient to support such a statement; 4 other prospective population studies were not included. For treatment there is no clear advice, just that treatment further reduces risk. However, the literature mentions IV(!) treatment of EM during pregnancy and shows that oral treatment (as given to non-pregnant women) with 500mg 3x/day Amoxicillin for 14-30 days is insufficient. The ‘pharmacokinetics’ during pregnancy is influenced differently depending on the type of antibiotics. No gynecologists took part in the guideline formation. Based on 4 case studies (1983-1988) is stated that ‘only seldom spirochetes are found’. However, in the nineties, only 5 more case-studies are found and these are not included. For several reasons the mother can have LD and test negatively and have low inflammation. To know whether a baby has been infected, tissue testing and repeated blots are needed to see if bands change. Damage depends on the trimester of transmission and no ‘syndrome’ has been described yet. During lactation L1 and L2 antibiotics can be used. German and American guidelines hardly mention pregnancy.

The literature confirms that Lyme Disease is transmissible. However, the discussion about the chance of transfer is influenced by numerous medical-scientific factors. How literature and factors have been incorporated in guidelines (Dutch CBO, IDSA, ILADS, DBG) is rather cursory. The Dutch guideline states that congenital Lyme is ‘seldom’ instead of ‘seldom investigated’ which arises a circular ‘evidence-based’ reasoning, that leads to the situation that necessary investigations are not done and congenital cases are not detected.

The Empty Sella Syndrome and Neuroborreliosis

Soutabh Vellala, BS

Background: Previous studies have associated peripheral facial nerve palsies, neuro radiculitis, meningeal signs etc. with confirmed neuroborreliosis. However, in its early stages, the condition is often overlooked. With the emergence of the novel coronavirus, understanding the ill-defined inflammatory symptoms is a priority.

Aims: This study was designed to elucidate any novel correlation between early through late-stage neuroborreliosis and an empty sella sign in patients with clear neurological symptoms.

Patients and Methods: Retrospective observational studies were conducted on 167 patients with a history of Neuroborreliosis acquired from August 2017 until August 2019. They were primarily taken and isolated if they had a 3-tesla unit MRI of the orbits, cavernous sinus, and/or internal auditory canal taken, lateral diplopia (H49.20), orthogonal diplopia (H49.10), and Argyll Robertson pupil (H57.01). Geographically, patient data was predominantly taken from a Lyme endemic area of Maryland and analyzed by multiple radiologists. On-going analysis is being done to cross-correlate patients with COVID-19. P-values were then obtained by comparing positive neuroborreliosis against empty sella appearances, lateral diplopia, and orthogonal gaze diplopia.

Results: Out of 167 patients, 86 had an MRI taken and 68 of these had an empty sella. Out of 77 patients who exhibited Argyll Robertson pupil, 60 had an empty sella. Additionally, out of 80 patients with lateral-gaze diplopia (H49.20), 65 had an empty sella. Finally, out of 70 patients with orthogonal-gaze diplopia, 58 had an empty sella. The data for patients with COVID-19 and Lyme disease is currently being analyzed. In comparing positive and negative t-tests for Argyll Robertson pupil, orthogonal gaze diplopia, and lateral gaze diplopia with an empty sella, a statistically significant correlation with a p<.0001 came across all comparisons.

Discussion: An empty sella, simply defined as fluid sitting in the subarachnoid space adjacent to the cavernous sinus, was found to be evident in patients with neuroborreliosis. The statistically significant value leads to a radiological insight on an empty sella. On normal MRI images of this space, there is a negligible amount of cerebrospinal fluid (CSF). However, when the subarachnoid space is filled, the pituitary appears crescent-shaped or absent, hence the term “empty sella”. Patients with a neurological manifestation of Lyme had statistically significant cases of an empty sella presumably caused by an increase in intracranial pressure. Not all cases returned positively for an empty sella, however, the statistical significance reaffirms its potential as a clinical tool to diagnose Neuroborreliosis.

COVID-19: Summary of Findings of Rapid Research Reviews in CAM for WHO

Paul Saunders, PhD, ND, DHANP, CCH

On March 30, 2020, the Complementary and Integrative Unit of the World Health Organization held an online meeting involving five world regions and eleven invited federations to discuss clinical experience, outcomes, and needs to address the COVID-19 pandemic. One outcome of that meeting was the need for rapid reviews on the safety, efficacy and risks of diverse alternative medicine treatments. The World Naturopathic Federation held a subsequent meeting across five health regions and identified clinical topics where rapid reviews could inform policy, practice, efficacy, and safety with respect to COVID-19 treatment. These treatments included NAC, vitamin C, vitamin D, Echinacea, Glycyrrhiza, Sambucus, Hedra, zinc, quercetin, honey, essential oils, selenium, resveratrol, homeopathy, micronutrients and several others. Several of these reports are completed and the rest will be done by early June 2020 for presentation to the World Health Organization. This presentation will provide a summary of findings in order to help clinicians make informed clinical treatment decisions.

The Role of Dental Toxicities in Chronic Disease

Stuart Nunnally, DDS, MS, NMD

Background: Dental toxins in the form of heavy metals such as mercury, or endotoxins from infected root canal treated teeth and jawbone osteonecrosis, pose immune challenges.

Aims: The current study demonstrates the prevalence and toxicity of heavy metals used in dentistry as well as the pathogenic organisms and their toxins found in root canal treated teeth and in jawbone osteonecrosis.

Patients and Methods: An updated review of the literature will be reported regarding heavy metal exposure from dentistry and its potential impact on Lyme patients. The presenter will report on his published data in the Journal of Orthomolecular Medicine, November 3, 2012 regarding root canal toxicity and his current DNA research on root canal treated teeth.

Results: The use of dental materials that contain mercury can be quantified using the Tri Test from Quicksilver Scientific. By testing blood, hair and urine, the various forms of mercury are speciated and quanitifice thereby revealing body burden and one’s ability to detoxify. The presenter’s current DNA study on root canal treated teeth corroborates his 2012 published paper in confirming that all root canal teeth are infected. The study analyzed extracted root canal teeth from twelve consecutive patients. Each patient presented with an infected root canal treated tooth as confirmed by Cone Beam Computed Tomography (CBCT) and a “healthy” root canal treated tooth. Although at the time of this submission, all of the data has not been tabulated, the evidence demonstrates that all root canal teeth are infected and that there is no difference in pathogenicity between a root canal treated tooth that is confirmed as infected by CBCT and one that appears healthy both clinically and by CBCT imaginaging.

Conclusions: Dental toxicities pose an immune challenge. Especially vulnerable to dental toxicities are those patients with chronic disease, including Lyme

Prevalence of Lyme Borreliosis Among TB Patients of Ternopil Region (Western Ukraine)

Mariia I. Shkilna, MD, PhD

Background: Tuberculosis (TB) continues to be a global problem for humanity. The effectiveness of TB treatment is associated not only with adequate etiotropic therapy, but also with the presence of concomitant diseases, including infectious etiology. Numerous studies of the features of the clinical course of Lyme disease indicate that LB is characterized by many symptoms similar to tuberculosis.
Research Aims/Objectives: The aim of the work was to identify the prevalence of Lyme borreliosis among patients with tuberculosis. Under the supervision were 99 patients with tuberculosis (TB) treated at the Ternopil Regional TB Dispensary during 2015-2017. The age of tested persons ranged from 18–76years. The group of examined patients comprised 77 men and 22 women.

Methodology: The screening test Elisa (Euroimmun) has been carried among all persons from the tested group towards the presence of antibodies IgM/IgG anti-Borrelia. Among those who met positive or doubtful results from screening test Elisa, another test of Western blot (Wb, Euroimmun) has been carried in order to confirm the infection. The persons tested were also questioned about the history of tick bites and Lyme- borreliosis.

Results/Findings: Antibodies of at least one IgM and / or IgG class to B. burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii and B. garinii) were detected in 25.2% of the examined patients with TB. Surveyed patients who indicated tick-borne bites, sero-confirmation of borreliosis was obtained in 9 out of 53 (17.0%), among 46 patients with tuberculosis who did not mind tick bites - 16 (34.7%) (p<0.05). Patients with tuberculosis with concomitant LB significantly more often noted delayed healing of cavities of decay in (36.0 ± 9,6)% of cases in 2 months of treatment compared with (14.9 ± 4,1)% of patients without it and cardiogram disoders (sinus bradycardia, conduction retardation and repolarization abnormalities) (p <0.05).

Conclusions: The prevalence of Lyme borreliosis in patients with tuberculosis is 25.2%. Concomitant Lyme borreliosis negatively affects the course of tuberculosis, which manifests itself in the slowed healing of cavities of decay and ECG disorders in this category of patients (p <0.05).

Multiplexed Paper-Based Test for Early Stage Lyme Disease

Zachary Ballard, PhD

Background- Diagnosing Lyme and associated tick-borne diseases at the point-of-care remains a major challenge. This is especially true during the early stages of the infection when many patients seek care and when treatment can be most effective. The gold-standard two-tier serological method for diagnosing Lyme disease suffers from high-cost, slow turn-around time, and seldom exceeds 50% sensitivity for early-stage samples, ultimately limiting access to antibiotic treatment, especially for early-stage patients without Erythema Migrans.

Aims- To address these challenges, we developed a rapid and inexpensive test as well as a serodiagnostic algorithm for diagnosing Lyme disease at the point-of-care. This study aimed to investigate the diagnostic performance of our newly developed assay.

Methods- Our point-of-care test comprises a paper-based vertical flow assay and a custom-designed mobile-phone reader to detect IgM and IgG antibodies using seven different Borrellia antigens and a synthetic peptide. Using a set of patient sera, we trained a neural network to infer a positive or negative result from the panel of antibody measurements as well as to select an optimal subset of antigen and peptide targets required for accurate diagnoses. We then blindly tested our assay and serodiagnostic algorithm using early-stage human samples (NPOS = 42, NNEG=54) from both the Midwest and Northeast regions of the United States, provided by the Lyme Disease Biobank.

Results- Using our machine-learning based approach we selected an optimal detection panel composed of P41 and OspC antigens for IgM detection, and P41, BmpA, Crasp1, along with a synthetic peptide (a C6-like epitope linked to a specific p41 epitope) for IgG detection. We achieved an AUC (area under the curve), sensitivity, and specificity of 0.95, 90.5%, and 87%, respectively. Implementing a batch-specific standardization and threshold tuning, we further achieved an AUC, sensitivity, and specificity of 0.963, 85.7%, and 96.3%, respectively.

Conclusions- This clinical study established the feasibility of a rapid (15 minutes) and low-cost (< $1 per test) multi-antigen test for diagnosing early-stage Lyme disease at the point of care. Our reported diagnostic performance exceeds that of what is commonly reported for two-tier testing during the early stage of the infection, showing promise as a point-of-care test for early-stage Lyme disease diagnosis.

Effective Killing of Borrelia burgdorferi in vitro with Novel Herbal Compounds

Jocelyn Strand, ND - speaker sponsored by Biocidin

Introduction: The tick-borne disease Lyme Borreliosis is caused by Borrelia bacteria. The disease can persist even after treatment with antibiotics, which is why other methods of treatment are needed. Herbal compounds and phytochemicals have been recently examined in relation to eradicating Borrelia bacteria in vitro.

Objective: The possible antimicrobial effect of two novel compounds, Biocidin£ Liquid and LSF Broad-Spectrum Liposomal formulas, was examined in the hopes of discovering an alternative method for eradication of Borrelia bacteria.

Methods: The minimum inhibitory concentrations (MICs) and minimum bacterial deaths (MBDs), as well as, time-kill effect of each compound were utilized in the study.

Results: The Liquid formula effectively killed the spirochetes with 1:10 dilution, while the MIC for the Liposomal formula was 1:25. Moreover, the MIC for both compounds with Round Bodies was 1:50 and for biofilms 1:10. Tough long-term effect (MBD) was seen only with 1:5 dilutions for both formulas. Additionally, the killing effect of each compound was observed already at 10 min post-treatment.

Conclusion: The study conducted here provides new insight into the antimicrobial effect of herbal compounds. Furthermore, studies such as these are required in order to discover possible alternatives to antibiotics in the battle against Borrelia infections.

Making Rational Choices when Testing for Tick-Borne Diseases

Joseph J. Burrascano Jr., MD - speaker sponsored by IGeneX

Clinicians want guidance in choosing which tests to run and from which labs. This choice should be based upon results of objective validation and proficiency test results, and publications. Labs that do not provide this data should raise concerns.

DIVERSITY OF TICK-BORNE PATHOGENS- Need to test for co-infections
In a 2018 study that tested ticks submitted by patients to Igenex, 11.3% contained Lyme Borrelia, 3.4% contained tick-borne relapsing fever (TBRF) Borrelia, 3.3% contained Rickettsia, 2.4% contained Babesia, 2.1% contained Ehrlichia and 0.4% contained Bartonella. A concurrent study in which over 10,000 patient samples tested by Igenex were reported, 37.3% were positive for Babesia species, 32.1% for Lyme Borrelia, 27.7% for TBRF, 19.1% for Bartonella, 16.7% for Anaplasma, 12.8% for Rickettsia and 6.9% for Ehrlichia. Positives were found in patient specimens from nearly every state.

Igenex immunoblots for Lyme Borrelia are able to detect antibodies to eight distinct strains and species, and when tested using validation samples from the CDC, New York, California, the CAP and NY Biologicals, sensitivity was 100% when interpreted with Igenex interpretative criteria, and 89% with CDC criteria. Specificity was 97% when using Igenex criteria and 99.3% when using CDC criteria. Because not every patient generates detectable levels of antibody, adding PCR testing in this cohort can increase disease detection by 12% (294 specimens tested).

Igenex immunoblots for TBRF are able to detect antibodies to six distinct species of TBRF. In well-characterized, PCR-positive patient samples, sensitivity was 100% (convalescent serum) and testing using a wide variety of specimen types revealed 97% specificity.

Igenex FISH testing detects circulating RNA of this organism at the genus level, meaning that it can pick up at least B. microti and B. duncani. Patient samples were analyzed by PCR at the NYS department of health, and all positive FISH tests were positive by their PCR. Igenex offers IFA testing for B. microti and B. duncani. Here too all (100%) of the positive IFAs were positive by PCR at the NYS dept. of health. Because not all Babesia patients have circulating parasite RNA, adding IFAs to the FISH testing increases positive yield by 12.4%. DNA PCR testing is also available.

Igenex offers a multispecies Bartonella western blot prepared from lysates of four species of this organism. Based on a study performed on 63 well-characterized clinical samples, 32 samples confirmed as being positive for antibodies to Bartonella and 31 samples negative for Bartonella but positive for antibodies to other tick-borne pathogens, the sensitivity of the Bartonella Western Blot was 97% and the specificity was 100%. Other Bartonella testing methods available include IFA, PCR, FISH and ELISPOT (detects T-cell reactivity to Bartonella).

Thanks to advancements in technology, testing quality has improved over the years. However, approach these new technologies cautiously if data on test accuracy is not provided.

Immune Regulation in the Age of COVID-19 and Tick-Borne Disease

Debby Hamilton, MD, MPH - speaker sponsored by Researched Nutritionals

Exposure does not equal infection. With the onset of COVID-19, it is evident that a proportion of people exposed will not become ill and this proportion is higher in children. It is also widely believed that with Lyme disease caused by the Borrelia bacteria, even with exposure not all people develop severe chronic disease. A seroprevalence study in Europe found similar levels of both IgG and IgM antibodies in people with symptoms of Lyme disease versus people with outdoor exposure but no symptoms. (Busova) It is important to study possible immune and inflammatory factors that may predispose one person to have symptoms where another is asymptomatic. In addition, not all people with acute Lyme develop chronic Lyme. Immune factors such as elevated CCL19 have been found in people who develop post treatment Lyme disease versus those that do not. (Aucott)

With COVID-19, estimates of asymptomatic carriers have been up to 30% with higher rates in children.(Gao) While most children are asymptomatic or have mild disease, there is a subset of children who develop an autoimmune Kawasaki like illness and researching who is at risk for this complication is important. We know age along with cardiovascular disease, hypertension and diabetes are all associated with increased risk from COVID-19. These conditions are associated with chronic inflammation and immune dysregulation.

The goal of this presentation is to determine from an immune and inflammatory standpoint who might be at higher risk for disease and morbidity from these illnesses. Once we understand who is at risk, then treatment can focus on balancing the immune system to increase the Th1 response needed to fight viruses and Borrelia. With autoimmune patients this might also include decreasing the inflammatory Th17 and Th2 responses to help support the Th1 response. Treatment also needs to focus on decreasing chronic inflammation to decrease severity of infections, especially COVID-19 which is associated with an abnormal late immune reaction leading to acute cytotoxic inflammatory responses.

Health System Resilience and COVID-19

CAPT Paul Reed, MD

The Health System Resilience Task Force, under the National Response Coordination Center (NRCC) for the current COVID-19 Public Health Emergency, focused on ensuring situational awareness and effective capacity to deliver COVID-19 care across the entire health system. Through its deliberations, pervasive impacts to health system access for non-COVID conditions were identified. Many direct as well as potential second and third order repercussions subsequently need to be understood, as these have untold implications with respect to health system resilience. It is clear the impact is likely to be felt for the foreseeable future, not just acutely during the pandemic. In consideration of this disruption to normal health system access the HHS Office of the Assistant Secretary for Health issued a Request for Information (RFI) on Health System Resilience, early in June. A robust response was received from a variety of organizations, providing valuable and diverse insights.

A data-informed, comprehensive analysis needed to be brought together to inform policy and action. This effort came to be known as the HHS Integrated Health System Analytics Enterprise (IHSAE). The concept for IHSAE is to develop various cohorts of Subject Matter Experts (SMEs) around aspects of health impacted by COVID-19 that could advise HHS on salient questions to investigate and the data necessary to inform analyses.

The ultimate goal of this enterprise is to develop a comprehensive, data-enabled analytic approach to assess the impacts of COVID-19 on timely access to quality health care and resultant health outcomes for all conditions, in order to inform immediate interventions and strategic planning that may ultimately guide resource allocation

IHSAE currently focuses on three use-case priorities in the context of COVID-19:

  1. Access to Care for Opioid Use Disorder, Mental Health Conditions and Behavioral Health Issues
  2. Access to Care for Elderly and Racial/Ethnic Minorities
  3. Diminished vaccination rates for children


What are some of the key insights from the ILADS Conference that the audience believes could be of value to address Health System Resilience, broadly and with respect to social determinants of health?

What insights from the ILADS Conference might help to inform policy decisions to improve access to care for elderly and minority populations, suffering disproportionately?

What insights from the ILADS Conference might benefit policy decisions to address increase in anxiety, depression, and suicide risk, increased during COVID-19?