Xenodiagnosis After Antibiotic Treatment for Lyme Disease - Phase II Study
Phase: NA
Conditions: Lyme Disease
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

 
 
 
 
 
 
Xenodiagnosis after Antibiotic Treatment for Lyme Disease – Phase  
2 Study 
 
 
Short Title: Lyme Xenodiagnosis Phase 2 Study 
 
 
Protocol Number: 15-I-0131 
Version 10  
Date: 22 March 2021 
 
 
Sponsored by: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
 
Investigational Device Exemption (IDE) # G150064 
 
 
Lead Principal Investigators (PIs): 
Adriana Marques, MD 
Linden Hu, MD 
 
Site PIs: 
Adriana Marques, MD (Laboratory of Clinical Immunology and Microbiology [LCIM]/NIAID) 
Linden Hu, MD (Tufts University) 
Gary Wormser, MD (New York Medical College [NYMC]) 
Kenneth Dardick, MD (Mansfield Family Practice) 
Luis Marcos Raymundo, MD (Stony Brook University) 
 
Study Sites: 
NIH Clinical Center 
Tufts Medical Center 
NYMC 
Mansfield Family Practice 
Stony Brook University (State University of New York)  
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
2
Sponsor Medical Monitor (SMM): 
Shirley Jankelevich, MD 
Medical Monitor 
Clinical Monitoring Research Program 
Leidos Biomedical Research, Inc. 
5705 Industry Lane, Room 248  
Frederick, MD 21704 
Phone 301-846-7322 
Email: shirley.jankelevich@nih.gov 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
3
 
TABLE OF CONTENTS 
 
TABLE OF ABBREVIATIONS .................................................................................................... 6 
STATEMENT OF COMPLIANCE ................................................................................................ 7 
PRÉCIS ........................................................................................................................................... 8 
1 
BACKGROUND .................................................................................................................... 9 
2 
STUDY OBJECTIVES ......................................................................................................... 11 
3 
STUDY DESIGN AND METHODS .................................................................................... 11 
3.1 Experimental Design ......................................................................................................... 11 
3.2 Inclusion Criteria ............................................................................................................... 12 
3.3 Exclusion Criteria .............................................................................................................. 13 
3.4 Exclusion from Skin Biopsy Part of the Protocol .............................................................. 14 
3.5 Special Populations ............................................................................................................ 14 
4 
STUDY PROCEDURES ...................................................................................................... 14 
4.1 Initial Visit ......................................................................................................................... 14 
4.2 Tick Placement Visit ......................................................................................................... 15 
4.3 Tick Removal (4-6 days after tick placement) .................................................................. 16 
4.4 1-Month and 3-Month Follow-up ...................................................................................... 17 
4.5 12 Months after Start of Antibiotic Therapy ..................................................................... 17 
4.6 Early Discontinuation/Withdrawn from the Study ............................................................ 17 
4.7 Repeat of Xenodiagnosis Procedure .................................................................................. 18 
4.8 Ticks .................................................................................................................................. 19 
4.9 Testing of the Xenodiagnostic Ticks ................................................................................. 21 
4.10 Optional Skin Biopsy ......................................................................................................... 21 
4.11 Discussion of Results ......................................................................................................... 21 
5 
RISKS ................................................................................................................................... 21 
5.1 Xenodiagnosis ................................................................................................................... 21 
5.2 Blood Drawing .................................................................................................................. 24 
5.3 Punch Skin Biopsy ............................................................................................................. 24 
6 
BENEFITS ............................................................................................................................ 24 
7 
STUDY ANALYSIS ............................................................................................................ 24 
7.1 Sample Size Justification ................................................................................................... 24 
7.2 Anticipated Difficulties ..................................................................................................... 26 
7.3 Interpretation and Impact ................................................................................................... 27 
8 
ASSESSMENT OF SAFETY ............................................................................................... 27 
8.1 Documenting, Recording, and Reporting Adverse Events ................................................ 27 
8.2 Definitions ......................................................................................................................... 27 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
4
8.3 Investigator Evaluation Adverse Events and Adverse Device Effects .............................. 29 
8.3.1 Severity ..................................................................................................................... 29 
8.3.2 Causality ................................................................................................................... 30 
8.4 Reporting Serious Adverse Events, Unanticipated Adverse Device Effects, and 
Unanticipated Problems to the IDE Sponsor ..................................................................... 31 
8.4.1 Adverse Events ......................................................................................................... 31 
8.4.2 Serious Adverse Events ............................................................................................ 31 
8.4.3 Unanticipated Problems ............................................................................................ 31 
8.4.4 Unanticipated Adverse Device Effects (UADEs) ..................................................... 31 
8.5 Follow-up of Adverse Events and Adverse Device Effects .............................................. 32 
8.6 Pausing Rules for the Protocol .......................................................................................... 32 
9 
DATA AND SAFETY MONITORING PLAN ................................................................... 32 
9.1.1 Safety Review and Communications Plan ................................................................ 33 
9.1.2 Sponsor Medical Monitor ......................................................................................... 33 
10 REPORTING PROCEDURES ............................................................................................. 33 
10.1 Investigator Reporting Responsibilities to the IRB ........................................................... 33 
10.1.1 Reporting Procedures at the NIH .............................................................................. 33 
10.1.2 Investigator Reporting Responsibilities to Local IRB(s) .......................................... 33 
10.2 Reporting to the NIAID Clinical Director ......................................................................... 33 
11 FINANCIAL COMPENSATION ......................................................................................... 33 
12 PLAN FOR MAINTAINING THE PRIVACY AND CONFIDENTIALITY OF SUBJECT 
RECORDS ............................................................................................................................ 34 
13 INTENDED USE OF THE SAMPLES/SPECIMENS/DATA ............................................. 34 
14 DATA MANAGEMENT PLAN .......................................................................................... 35 
15 SUBJECT RECRUITMENT ................................................................................................ 36 
16 CONSENT PROCEDURES ................................................................................................. 37 
17 STUDY RECORDS RETENTION ...................................................................................... 37 
18 PROTOCOL MONITORING ............................................................................................... 37 
19 CERTIFICATE OF CONFIDENTIALITY .......................................................................... 38 
REFERENCES ............................................................................................................................. 39 
APPENDIX 1: FOLLOWING PUNCH BIOPSY OF THE SKIN ............................................... 44 
APPENDIX 2: CARE OF THE XENODIAGNOSIS SITE ......................................................... 45 
APPENDIX 3: TOXICITY GRADING SCALE .......................................................................... 46 
 
LIST OF TABLES 
Table 1: Maximum Number of Xenodiagnosis Procedures to be Offered ................................... 18 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
5
Table 2: Sample Size Needed for 80% Power with a Type I Error Rate of 0.05, and Varying 
Assumptions for the True Prevalence Rates for Asymptomatics (p1) and Symptomatics 
(p2) ................................................................................................................................. 25 
Table 3: Exact 95% Confidence Interval for the Observed Positivity Rate, for Varying Group 
Size (N) and Observed Numbers of Positive Individuals .............................................. 25 
Table 4: Financial Compensation ................................................................................................. 34 
 
 
LIST OF FIGURES 
Figure 1: Feeding of Ixodes Larvae under LeFlap Dressing......................................................... 15 
 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
6
TABLE OF ABBREVIATIONS 
 
ADE 
adverse device effects  
AE 
adverse event 
BTPV 
blacklegged tick phlebovirus 
CDC 
US Centers for Disease Control and Prevention 
CRFs 
case report forms  
CFR 
Code of Federal Regulations 
CSO 
Clinical Safety Office 
ELISA 
enzyme-linked immunosorbent assay 
EM 
erythema migrans  
FDA 
US Food and Drug Administration 
FNLCR 
Frederick National Laboratory for Cancer Research  
GCP 
Good Clinical Practice 
HRPP 
Human Research Protections Program 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
IDE 
Investigational Device Exemption 
IRB 
Institutional Review Board  
LCIM 
Laboratory of Clinical Immunology and Microbiology 
MM 
Medical Monitor  
NIAID 
National Institute of Allergy and Infectious Diseases  
NIH 
National Institutes of Health 
NYMC 
New York Medical College 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations  
OHRP 
Office for Human Research Protections  
PCR 
polymerase chain reaction 
PI 
Principal Investigator  
PLDS 
post-Lyme disease syndromes  
PTLDS 
post-treatment Lyme disease syndrome  
SAEs 
serious adverse events 
SBV 
South Bay virus 
SCID 
Severe Combined Immunodeficiency 
SMM 
Sponsor Medical Monitor 
SRCP 
Safety Review and Communication Plan 
UADEs 
unanticipated adverse device effects  
UPs 
unanticipated problems 
UPnonAE 
unanticipated problem that is not an adverse event 
US 
United States 
 
 
 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
7
STATEMENT OF COMPLIANCE 
 
The trial will be carried out in accordance with International Council for Harmonisation of 
Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice 
(GCP) and the following:  
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies (45 
CFR Part 46, 21 CFR Part 50, 21 CFR Part 56, 21 CFR Part 312, and/or 21 CFR Part 812)  
 
NIH-funded investigators and clinical trial site staff who are responsible for the conduct, 
management, or oversight of NIH-funded clinical trials have completed Human Subjects 
Protection and ICH GCP Training. 
 
The protocol, informed consent form(s), recruitment materials, and all participant materials will 
be submitted to the Institutional Review Board (IRB) for review and approval. Approval of both 
the protocol and the consent form must be obtained before any participant is enrolled. Any 
amendment to the protocol will require review and approval by the IRB before the changes are 
implemented to the study. In addition, all changes to the consent form will be IRB-approved; an 
IRB determination will be made regarding whether a new consent needs to be obtained from 
participants who provided consent using a previously approved consent form. 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
8
PRÉCIS 
 
Lyme disease is the most common vector borne disease in the United States. Although antibiotic 
therapy is clinically effective in treating the symptoms of Lyme disease for most patients early in 
the course of disease, a significant number of patients who receive therapy report persistent 
symptoms. The cause of persistent symptoms after antibiotic therapy for Lyme disease is an area 
of great controversy. Recent studies have shown that the organism (Borrelia burgdorferi) may 
persist in animals after antibiotic therapy and can be detected by using the natural tick vector 
(Ixodes scapularis) to acquire the organism through feeding (xenodiagnosis). Whether this 
occurs in humans is unknown. Currently available tests for human Lyme disease do not allow 
determination of persistent infection after antibiotic therapy.  
 
We performed the first study of the use of I. scapularis larva for the xenodiagnosis of B. 
burgdorferi infection in humans. Our pilot study showed that xenodiagnosis was well tolerated 
with no severe adverse events (AEs). The most common AE was mild itching at the site. In this 
small pilot study, xenodiagnosis for B. burgdorferi was positive in 2 participants and 
indeterminate in 2 participants. Further studies are needed to determine the sensitivity of 
xenodiagnosis in evaluating the infection status of Lyme disease patients.  
 
In this proposal, we want to further investigate the utility of xenodiagnosis for identifying 
persistence of infection with B. burgdorferi in treated human Lyme disease. Our objectives 
include assessing the link between detection of B. burgdorferi by xenodiagnosis and persistence 
of symptoms in patients diagnosed with Lyme disease, within 1 year, post therapy; compare the 
rate of detection of B. burgdorferi by xenodiagnosis after therapy in participants with post-
treatment Lyme disease symptoms; identify subject characteristics related to the likelihood of 
detecting B. burgdorferi by xenodiagnosis including: time from infection, time between infection 
and therapy, time from therapy; and continue to assess the safety of xenodiagnosis in humans.  
 
The results of study have the potential to resolve this long-standing controversy in Lyme disease 
pathogenesis. While xenodiagnosis is unlikely to be widely used in clinical practice due to the 
labor intensity and speed of testing, if our study shows a linkage between positive 
xenodiagnostic testing and persistence of symptoms after B. burgdorferi infection, it may prove 
to be a useful tool for testing new strategies for treatment and for correlation with more generally 
applicable diagnostic markers. Understanding the pathogenesis of persistent symptoms following 
Lyme disease, and identifying reliable diagnostic tests for determining the success of antibiotic 
therapy, is critical to the medical management of these patients.  
 
 
 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
9
1 
BACKGROUND 
 
Lyme disease is the most common vector borne disease in the United States 1. It is transmitted to 
humans via the bite of infected Ixodes ticks. At the tick bite site, Borrelia burgdorferi, the 
causative organism of Lyme disease, enters the skin, often resulting in the characteristic 
erythema migrans (EM) rash. From the original entry site, the organism spreads to distant 
locations affecting the heart, joints and central nervous system. Many studies have shown that 
Lyme disease is treated successfully with antibiotics in the majority of cases, and patients with 
objective evidence of treatment failure are rare with currently recommended regimens 2-5. 
Patients with late manifestations can have a slower response to therapy, sometimes taking weeks 
or months to recover 6-14. 
 
A portion of patients treated for Lyme disease will have persistent or relapsing non-specific 
symptoms (such as fatigue, musculoskeletal pain, and cognitive complaints) after receiving an 
adequate course of antibiotic therapy, a condition that is called post-Lyme disease syndrome. 
The best estimates of the prevalence of post-Lyme disease syndrome come from studies of 
patients with erythema migrans who received appropriate antibiotic treatment. 10-40% of such 
patients have persistent or intermittent subjective symptoms of mild to moderate intensity 12 
months after completion of therapy 3,4,15-19. The most common post-Lyme disease symptoms are 
fatigue, arthralgias, myalgias, headache, neck stiffness, paresthesias, sleeplessness, irritability, 
and difficulty with memory, word finding, and concentration 3,4,15-18. The appearance of post-
Lyme disease symptoms seems to correlate with disseminated disease, a greater severity of 
illness at presentation, and delayed antibiotic therapy 3,20-24. The pathogenesis of these symptoms 
(whether due to persistent infection, immunologic dysfunction, or other mechanism) remains an 
area of great controversy.  
 
A major concern has been that the symptoms of post-Lyme disease syndrome may represent 
persistent infection with B. burgdorferi. Hunfeld et al. 25 reported a 50% recovery rate of B. 
burgdorferi from erythema migrans skin biopsies done before therapy in 3,421 patients, and 
1.7% of skin biopsies of patients (19 out of 1148) remained B. burgdorferi positive by culture 2 
months (range 1.3-3 months) after antibiotic therapy. Spirochete persistence was not accounted 
for by the development of resistance to the antibiotic. Objective evidence of Borrelia infection in 
patients with post-Lyme disease syndrome has not been found using polymerase chain reaction 
(PCR) 26,27 or culture 26,27. It should be noted, however, that B. burgdorferi culture and PCR have 
low sensitivity in most body fluids from patients with Lyme disease28,29. Other tests that have not 
been helpful in evaluating patients with post-Lyme disease syndrome include changes in C6 
antibody levels 30 and antibodies in immune complexes 31. Current serologic testing for 
antibodies to B. burgdorferi remains positive for years after therapy and declines very slowly 
making it essentially useless for confirmation of successful therapy. There are many anecdotal 
reports of patients with different persistent symptoms improving with prolonged antibiotic 
therapy (and worsening with cessation), but randomized controlled trials to date have not shown 
significant, sustained benefit from antibiotic therapy 26,27,32. The question of whether patients 
continue to harbor B. burgdorferi after antibiotic therapy has become a very contentious issue33.  
 
Although the prevailing belief in the scientific and medical establishments is that patients do not 
continue to harbor B. burgdorferi and that long-term antibiotics are of no value, recent animal 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
10
data suggest that eradication of B. burgdorferi by antibiotics may not be complete. Studies in 
dogs, mice and non-human primate have shown that B. burgdorferi DNA can be detected in 
tissues for extended periods after antibiotic therapy34-40. Though positive testing for B. 
burgdorferi DNA by PCR has been used for diagnosis of the infection 41 42 43,44, it must be noted 
that PCR has the potential to recognize DNA from dead or dying bacteria 45. 
 
Obtaining tissue samples of heart, joint, or bladder for testing would not be feasible as a 
diagnostic test in humans. Ixodes ticks feeding on antibiotic treated animals were able to acquire 
B. burgdorferi during their blood meal 34. The organism could be detected by PCR of the tick 
and could also be transmitted to immunodeficient SCID mice by the ticks during their next blood 
meal. These studies have now been replicated by other groups both in mice and in monkeys. 38-
40,46,47 
 
The use of a natural vector (i.e., I. scapularis) to detect the presence of an organism is called 
xenodiagnosis. Xenodiagnosis has been used in the diagnosis of human infections in instances 
where other diagnostic testing is difficult either due to difficulties/risks of obtaining proper 
specimens for testing or due to difficulty in cultivating the organism. Xenodiagnosis has long 
been used in Lyme disease research to provide definitive evidence of a host's infection status. 
For Lyme disease, tick saliva has been shown to be a chemoattractant for the organism and thus 
feeding ticks have the potential to aggregate and concentrate bacteria from a wide area, 
improving sensitivity 48.  
 
Xenodiagnosis by feeding uninfected ticks on patients after antibiotic therapy could offer a 
minimally invasive method for determining the persistence of B. burgdorferi after antibiotic 
therapy. We have performed the first study of the use of I. scapularis larva for the xenodiagnosis 
of B. burgdorferi infection in humans. The primary goals of this study were to develop 
procedures that could be used in xenodiagnostic testing of patients after antibiotic therapy with 
Lyme disease and to determine the safety of tick xenodiagnosis in humans.  
 
We have performed xenodiagnosis in 43 individuals. Xenodiagnosis has been well tolerated with 
no severe AEs. The most common AE was mild itching at the tick attachment site. Results from 
the initial 36 participants are described in our manuscript “Xenodiagnosis to detect Borrelia 
burgdorferi infection: A first-in-human study”, which has been published in the Clinical 
Infectious Diseases journal49.  
 
Participants who underwent xenodiagnosis included 10 patients with high C6 antibody levels, 10 
patients with post-treatment Lyme disease syndrome (PTLDS), 5 patients with acute erythema 
migrans after completion of antibiotic therapy, one patient with erythema migrans on therapy 
(who also underwent xenodiagnosis after antibiotic therapy), and 10 healthy volunteers. Seven 
patients underwent more than one xenodiagnosis procedure.  
 
As no previous protocol for xenodiagnosis with I. scapularis larva in humans existed, we 
developed a retention dressing using the LeFlap™ (Monarch Labs, Irvine, CA) dressing, which 
was modified for use with ticks. Participants enrolled early in the trial had fewer ticks feed 
successfully due to issues with the tick retention dressing that resulted in entrapment of the ticks 
in the adhesive. 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
11
After modifying the dressing by adding a foam ring to create a barrier between ticks and 
adhesive, we were more consistently able to get between 30 and 50% of ticks to feed 
successfully.  
 
Xenodiagnosis was well tolerated. All participants successfully completed the tick placement and 
there were no withdrawals during the study. The most common AE was mild itching at the site, 
which was seen in 58% of participants, with a median duration of 3 days. Repeat of the 
xenodiagnosis procedure was similarly well tolerated, with mild itching at the site as the most 
common complaint. There were no serious AEs associated with the procedure. Although not a 
primary goal of this pilot study, we did find 2 participants that tested positive and 2 participants 
who tested indeterminate for the presence of B. burgdorferi in the xenodiagnostic ticks.  
 
In this Phase 2 study, we plan to expand our study of xenodiagnosis for Lyme disease in humans. 
Xenodiagnostic evidence that B. burgdorferi survives in humans after antibiotic therapy may 
fundamentally change the approach to patients with persistent symptoms and provide physicians 
and researchers with a new tool with which to study the mechanisms of disease.  
 
2 
STUDY OBJECTIVES 
 
1. Objective #1:  
Assess the link between detection of B. burgdorferi by xenodiagnosis and persistence of 
symptoms in patients diagnosed with Lyme disease, within 1 year, post therapy. 
 
2. Objective #2: 
Compare the rate of detection of B. burgdorferi by xenodiagnosis after therapy for Lyme 
disease in participants with symptoms of post-treatment Lyme disease. 
 
3. Objective #3: 
Identify subject characteristics related to the likelihood of detecting B. burgdorferi by 
xenodiagnosis including: time from infection, time between infection and therapy, time 
from therapy. 
 
4. Objective #4:  
Continue to assess the safety of xenodiagnosis in humans.  
 
3 
STUDY DESIGN AND METHODS 
3.1 
Experimental Design 
This will be a prospective non-randomized multi-site study conducted at 5 sites: 1) Tufts Medical 
Center, Boston, MA (Dr. Linden Hu, co-PI and Tufts site PI); 2) NIH Clinical Center, Bethesda, 
MD (Dr. Adriana Marques, co-PI and NIH site PI); 3) New York Medical College, Valhalla, NY 
(Dr. Gary Wormser, site PI) and 4) Mansfield Family Practice, Storrs, CT (Dr. Kenneth Dardick, 
site PI), and 5) Stony Brook University, Stony Brook, NY (Dr. Luis Marcos Raymundo, site PI). 
  
All investigators are well-recognized experts in the field of Lyme disease and have prior 
experience in research. Up to 240 participants will be enrolled across all sites. 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
12
3.2 
Inclusion Criteria 
Criteria for the diagnosis and therapy for Lyme disease can be found at “The clinical assessment, 
treatment, and prevention of Lyme disease, human granulocytic anaplasmosis, and babesiosis: 
clinical practice guidelines by the Infectious Diseases Society of America” 5. 
 
Patients with Lyme disease, post-therapy (N=100) 
1. Age 18 or older. 
2. Lyme disease diagnosed in the previous 13 months, fulfilling the case definition of confirmed 
or probable Lyme disease by the US Centers for Disease Control and Prevention (CDC)  
https://wwwn.cdc.gov/nndss/conditions/lyme-disease/case-definition/2017/ . 
3. Completion of 1 course of antibiotics at least 3 months and up to 12 months between the end 
of the therapy and the xenodiagnostic procedure. 
4. Antibiotic treatment fulfills the Infectious Diseases Society of America guidelines for the 
recommended therapy for Lyme disease 5.  
 
Patients with post-Lyme disease complaints at least 12 months from initial treatment (N=40) 
1. Age 18 or older. 
2. Diagnosed with confirmed or probable Lyme disease fulfilling the case definition of Lyme 
disease by the CDC  
https://wwwn.cdc.gov/nndss/conditions/lyme-disease/case-definition/2017/ . 
3. Received recommended antibiotic therapy for Lyme disease 5 at least 12 months between the 
end of the initial antibiotic therapy and the xenodiagnostic procedure. 
4. Persistent or recurrent symptoms that began or worsened within 6 months of the diagnosis 
and treatment for Lyme disease. 
 
Acute EM (N=40) 
1. Age 18 or older. 
2. EM diagnosed by the study physician.  
3. Receiving antibiotic therapy for Lyme disease for less than 48 hours. 
 
Lyme Arthritis (N=40) 
1. Age 18 or older. 
2. Lyme arthritis50 and have not received antibiotic therapy for the disease. 
 
Healthy volunteers (N=20) 
1. Age 18 or older. 
2. No prior history of Lyme disease. 
3. Negative whole cell enzyme-linked immunosorbent assay (ELISA) or C6-based antibody test 
for Lyme disease. 
 
Patients with recently diagnosed (acute) EM (within 48 hours of starting antibiotic therapy) and 
patients with untreated Lyme arthritis will be recruited in an attempt to increase the chances of 
finding a positive result by xenodiagnosis (an attempt of a “positive control”). While patients 
with acute untreated EM would be the best positive control group, it would be unethical to 
withhold therapy in these patients for the few days required for tick feeding, due to the risk of 
dissemination of the organism and possible morbidity. Patients with untreated Lyme arthritis will 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
13
be recruited to establish whether xenodiagnosis can be used to identify infection in late stage 
Lyme patients where the bacterium is known to be present. These patients have been infected for 
months and will not be harmed for delaying therapy for a few days. Lyme arthritis is a late 
manifestation of B. burgdorferi infection, and hematogenous dissemination already occurred at 
this late stage. Studies have shown that the presence or absence of previous antibiotic treatment 
is more predictive than the duration of untreated arthritis for the success of antibiotic therapy in 
Lyme arthritis. 6,8,51-53 Similarly, patients who just started therapy for EM may still have live 
Borrelia in the skin and xenodiagnosis may be able to recover the bacteria (but culture of skin 
biopsies from patients with EM become negative very quickly - within one dose - on antibiotic 
therapy).  
 
While treatment for Lyme disease will not be offered under this protocol, it may be available via 
different clinical research protocols or regular medical care at the study site. If not, treatment will 
be prescribed by the patient’s primary care. For patients with untreated early Lyme disease 
(erythema migrans), antibiotics can be started at the same day of tick placement. For patients 
with untreated Lyme arthritis, antibiotics can be started after collection of xenodiagnostic ticks 
(usually 4-5 days, up to 7 days). For patients with Lyme arthritis, if less than 14 ticks fed 
successfully and if the participant agrees, antibiotic treatment can be delayed until after the 
repeat procedure (see Section 4.7). 
 
Patients with acute EM and untreated Lyme arthritis will be able to re-enroll as Patients with 
Lyme disease, post-therapy. Therefore, in case of positive results, we will be able to compare 
between the procedures.  
 
Negative control patients will include healthy volunteers from Lyme endemic areas who have 
never been diagnosed with Lyme disease and have a negative B. burgdorferi ELISA and C6 
antibody titer. 
3.3 
Exclusion Criteria 
1. No antibiotic therapy active against Lyme disease in the previous 3 months (except patients 
with acute EM). Prophylaxis with a single dose of doxycycline 200 mg is not an exclusion. 
2. History of allergy to surgical tape or dressing.  
3. History of severe reactions to tick bites (granuloma or systemic reactions). 
4. Inability to maintain the dressing for any reason.  
5. Pregnancy or lactation. 
6. Unwillingness to use an effective method of birth control for the duration of participation in 
the study (women of child-bearing potential only) and for at least 3 months following the last 
tick placement. 
7. Use of investigational therapy and devices during the time of the study and/or in the month 
prior to signing the informed consent. 
8. Active severe skin disease, uncontrolled diabetes, cancer other than non-melanoma skin 
cancers, autoimmune disease requiring immunosuppressive therapy, or history of HIV, 
chronic viral hepatitis, or syphilis.  
9. Oral or IV steroids in the previous 2 weeks (topical, nasal, inhaled, intra-articular, and 
replacement doses of steroids are not exclusions). 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
14
10. Any other condition that, in the opinion of the investigator, would make the patient 
unsuitable for enrollment or could interfere with the patient participating in and completing 
the study. 
11. Refusal to participate in specimen collection and storage for future study related use. 
3.4 
Exclusion from Skin Biopsy Part of the Protocol 
1. History of forming large thick scars (keloids) after skin injuries or surgery. 
2. History of excessive bleeding after cuts or procedures.  
3. Currently taking anticoagulants.  
4. History of allergy to lidocaine. 
3.5 
Special Populations 
Children: Children are excluded from this protocol as there is no direct benefit to the 
participants, the risk from the skin biopsies is small but above minimal, the procedure is invasive 
and can be stressful for children, and there is concern over their ability to maintain the LeFlap 
dressing. 
 
Women: Pregnant and breastfeeding women are excluded from trial participation as there is no 
direct benefit to the participants. 
 
Adults who lack capacity to consent: Adults lacking decision-making capacity to provide 
informed consent are excluded at screening as there is no direct benefit to the participants. 
Enrolled participants who permanently lose the ability to consent during participation will be 
monitored for safety but will have no additional research procedures performed. 
 
NIH staff: NIH staff and family members of study team members may be enrolled in this study 
as this population meets the study entry criteria.  Neither participation nor refusal to participate 
as a subject in the research will have an effect, either beneficial or adverse, on the participant’s 
employment or position at NIH. 
Every effort will be made to protect participant information, but such information may be 
available in medical records and may be available to authorized users outside of the study team 
in both an identifiable an unidentifiable manner. 
The NIH Information Sheet on NIH Staff Research Participation will be made available.  Please 
see Section 16 for consent of NIH Staff. 
 
4 
STUDY PROCEDURES 
4.1 
Initial Visit 
After written informed consent is obtained, participants will have a history and a physical 
examination performed. Blood will be drawn and tested for Lyme serologies (approximately 8 
mL of blood). Patients with acute EM will also have blood collected for culture, PCR, and other 
direct tests for B. burgdorferi (approximately 50 mL). These tests may be performed at NYMC, 
NIH, and Tufts laboratories. This initial visit may or may not be combined with the tick 
placement visit (see below).  

 
 
Protocol Version 10 
22 Mar 2021 
 
 
15
4.2 
Tick Placement Visit 
This visit may occur together with the initial visit, or may be a separate visit, up to 1 month 
apart. If a separate visit, eligibility criteria will be reviewed again to ensure patient remains 
eligible. At this visit, approximately 40 mL of blood will be drawn and a urine sample may be 
collected and stored for future study related research. Stored specimens will be useful for future 
development of new diagnostic tests or to test for as yet to be identified emerging infectious 
agents. Female patients of childbearing potential will have a urine or blood pregnancy test 
performed at this visit. The results of the pregnancy test will be reviewed before the placement of 
the ticks. Self-reported symptoms will be characterized using symptom scales, the SF-36v2 
questionnaire, and fatigue scales. 
 
Except for patients with acute EM, ticks will not be placed at this visit if the individual is found 
to have abnormal vital signs (temperature >38.0°C, a pulse greater than 105 beats per minute, 
systolic blood pressure >160 mm/Hg or diastolic blood pressure >95 mm/Hg, in at least 2 
measurements) and these individuals will be asked to follow up with their personal physician. 
They may be re-evaluated for tick placement at a subsequent time when the vital signs are within 
acceptable limits. 
 
Skin Biopsy 
Eligible participants (acute EM, untreated Lyme arthritis and PTLDS) who agree to undergo the 
optional skin biopsy will have two 2 or 3 mm skin punch biopsies performed outside the site of 
the tick placement. The skin will be cleansed and anesthetized with 2 percent lidocaine with 
epinephrine. Each biopsy will be performed using a “punch”- a small disposable tubular knife. 
The wound will be closed, if necessary, with steri-strips or (rarely) with 1 or 2 interrupted nylon 
sutures. A bandage will be applied and participants will receive the instruction sheet regarding 
follow up procedures (Appendix 1). If suture(s) are placed, patients will be asked to return in 7 to 
10 days to have the suture(s) removed. Biopsies will not be done on face, neck, scalp, or over the 
tibia. One biopsy will be used for B. burgdorferi culture and/or PCR. A second biopsy will be 
stored for future study related research.  
 
If the Initial Visit and the Placement Visit do not occur together, the initial skin biopsy may be 
performed at either visit, accordingly to the preference of the participant and study personnel.  
 
Xenodiagnosis 
Between 25-30 larval ticks that have been aged and are known to be ready to attach will be 
placed (Figure 1). The area chosen for the placement will be as close as possible to the site of the 
previous EM, if possible. Ticks will not be attached to head, neck or genitalia. If necessary, body 
hair will be shaved at the site. The ticks will be placed inside the Creature Comforts II LeFlap™ 
dressing which has been modified for tick use with the addition of a foam inner layer. The 
LeFlap is a pre-assembled confinement dressing that is used with medical maggots. Participants 
will be requested to keep the dressing dry for the duration of the attachment. Participants will 
receive an instruction sheet with instructions regarding care of the dressing and a Diary Card. 
The subject will be asked to complete a diary card daily starting the date of placement of the 
ticks through 30 days after tick removal. 
 
Figure 1: Feeding of Ixodes Larvae under LeFlap Dressing 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
16
 
 
4.3 
Tick Removal (4-6 days after tick placement)  
Larval ticks typically feed for 4 to 5 days but it is possible that some might feed as long as 
10 days. Participants will return to the study clinic for removal of the dressing and recovery of 
the ticks at 4-6 days. Pictures may be taken of the site. Approximately 40 mL of blood will be 
drawn for future study related research. Participants will be asked during the visit whether there 
were any complications during the feeding of the ticks, including adverse reactions and tick 
escape, and the diary card will be reviewed at this visit. 
 
If there are a significant number of ticks still attached, the individual may be asked to return over 
the next subsequent days for evaluation until the majority of the ticks have detached. If this 
occurs, the ticks that dropped off will be collected, the dressing will be changed, and the 
individual will be seen within 24-72 hours for removal of the other ticks and completion of the 
visit. These visits may need to be repeated up to day 10 from the time of placement. 
 
Eligible participants who agree to undergo the optional skin biopsy will have two 2 or 3 mm skin 
punch biopsies performed outside the site of the tick placement. The skin will be cleansed and 
anesthetized with 2 percent lidocaine with epinephrine. Each biopsy will be performed using a 
“punch”- a small disposable tubular knife. The wound will be closed, if necessary, with steri-
strips or (rarely) with 1 or 2 interrupted nylon sutures. A bandage will be applied and participants 
will receive the instruction sheet regarding follow up procedures (Appendix 1). If suture(s) are 
placed, patients will be asked to return in 7 to 10 days to have the suture(s) removed. Biopsies 
will not be done on face, neck, scalp, or over the tibia. One biopsy will be used for B. 
burgdorferi culture and/or PCR. A second biopsy will be stored for future study related research.  
 
The study team will contact each subject 7-10 days post-tick removal to determine if they are 
experiencing any AEs. The participants will also be instructed to call the study staff if any new 
symptoms occur during the period when the ticks are attached and for 30 days after tick removal. 
A 30-day window was chosen for the following reasons: 1) Adverse reactions to tape or the tick 
bites are expected to resolve within 2 weeks after removal of the ticks; 2) in the very unlikely 
event that there is an as yet unidentified infection that is transmitted by the ticks, all of the known 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
17
tick transmitted diseases have an onset of symptoms within 30 days of tick detachment. 
Participants will be specifically told to contact the study personnel for any fever, chills, joint 
pains, muscle pains, neck stiffness, or severe headache, as these are symptoms of known tick-
borne diseases. For the first month after removal of the ticks, the participants will be asked to 
maintain a daily diary, which will be collected at the 1-month visit. 
4.4 
1-Month and 3-Month Follow-up 
In addition to the contact with the individual at 7-10 days post-tick removal, participants will be 
followed at 4 to 6 weeks after the removal of ticks to ensure that no unanticipated events have 
occurred. This visit may be in person or by phone. Participants will be specifically questioned 
about fever, rash or infection at the site of tick attachment. They will be questioned about any 
intercurrent illnesses. Self-reported symptoms will be characterized using symptom scales, the 
SF-36v2 questionnaire and fatigue scales. Forms are self-administered and can be administered 
in paper form or computer, including online. If the visit is in person, approximately 40 mL of 
blood will be drawn and a urine sample may be collected for storage for future study related 
research. The diary card will be reviewed at this visit. 
 
Participants will also be followed at 3 months (±2 weeks) after the mesh is removed. This visit 
may be in person or by phone. Participants will be questioned about any intercurrent illnesses. 
They will complete the symptom scales, the SF-36v2 questionnaire and fatigue scales. Forms are 
self-administered and can be administered in paper form or computer, including online. If the 
visit is in person, approximately 40 mL of blood will be drawn and a urine sample may be 
collected for storage for future study related research.  
 
Participants will be contacted to discuss their individual results from xenodiagnostic studies 
when these become finalized. The results are expected to be available within one year of the 
procedure. 
4.5 
12 Months after Start of Antibiotic Therapy 
Participants enrolled under the Lyme disease, post treatment group will also be contacted at 12 
months (±4 weeks) after the start of their antibiotic therapy for Lyme disease. This visit may be 
in person or by phone. If the visit is in person, approximately 40 mL of blood will be drawn and 
a urine sample may be collected for storage for future study related research. Self-reported 
symptoms will be characterized using symptom scales, the SF-36v2 questionnaire and fatigue 
scales. Forms are self-administered and can be administered in paper form or computer, 
including online. 
4.6 
Early Discontinuation/Withdrawn from the Study 
If a participant decides to withdraw from the study, the investigator may ask the participant who 
is withdrawing whether the participant wishes to provide continued follow-up and further data 
collection subsequent to their withdrawal from the interventional portion of the study.  
Participants that undergo tick placement and discontinue study participation by the PI’s wishes 
will continue to come to visits for subject safety assessments or may be asked to provide vital 
safety information over the phone. Attempts will be made to contact participants who are lost to 
follow up. 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
18
4.7 
Repeat of Xenodiagnosis Procedure 
 
All repeats of the xenodiagnosis procedure are optional. 
 
Repeat of the xenodiagnosis procedure can be offered to participants in 3 situations: 
a. Participants in whom the xenodiagnosis procedure failed to recover at least 14 intact 
engorged ticks. 
b. Participants in whom the xenodiagnosis procedure is positive. 
c. Participants who have completed the study as acute EM or Lyme arthritis patients and are 
eligible to participate as Patients with Lyme disease, post-therapy. 
 
For participants in whom at least 14 intact engorged ticks are not recovered, 1 additional 
xenodiagnosis procedure (placement of ticks) will be offered. This does not require re-
enrollment. Based on our experience with the phase 1 study, we expect that this may occur in up 
to 50% of the placements. Xenodiagnostic procedure will not be offered to participants with 
acute erythema migrans, as they will have started antibiotic therapy at the time of the initial tick 
placement.  
 
For participants in whom the xenodiagnosis procedure is positive, re-enrollment will likely be 
required, due to the time necessary for results from the testing of the xenodiagnostic ticks to be 
finalized, as participants will likely have completed the follow up phase of the study. The study 
procedures will be repeated as described above, including the skin biopsy (optional) and follow 
up visits.  
 
Participants who completed the study as Acute Erythema Migrans or Lyme Arthritis patients, and 
are eligible to participate as Patients with Lyme disease, post-therapy, will be enrolled as part of 
the new patient group and a new study number will be assigned. The study procedures will be 
repeated as described above, including the skin biopsies (optional) and all follow up visits. 
 
Therefore, the maximum number of xenodiagnosis procedures offered to a participant for each 
group and situation, and the expected time period for the procedures are described in the Table 1 
below.  
 
Table 1: Maximum Number of Xenodiagnosis Procedures to be Offered 
Group 
Description 
Xenodiagnosis, 
initial 
If xeno 
positive 
Enroll as a 
new 
participant? 
Total 
Time 
period 
between 
first and 
last 
possible 
tick 
placement 
1 
Patients with 
Lyme disease, 
post-therapy  
Up to 2 
Up to 2 
No 
Up to 4 
3-6 
months 
2 
Patients with post-
Lyme disease 
Up to 2 
Up to 2 
No 
Up to 4 
3-6 
months 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
19
complaints at least 
12 months from 
initial treatment 
3 
Acute EM  
1* 
* 
Yes, as 
Group 1 
Up to 5 (1 as 
EM, up to 4 as 
post-therapy) 
7-18 
months 
4 
Lyme Arthritis 
Up to 2 
* 
Yes, as 
Group 1 
Up to 6 (2 as 
LA, up to 4 as 
post-therapy) 
7-18 
months 
5 
Healthy volunteers  Up to 2 
 
No 
Up to 2 
2 weeks 
* Repeat will not be offered, as patients will have received antibiotic therapy at this point and 
results would not be comparable. 
 
The date of each visit will be determined by the date from each procedure. In case there is 
overlap of visits, visits may be combined. Participants will be followed until completion of the 
protocol from the time of the last xenodiagnosis procedure. Lyme serologies will be repeated if 
performed more than 2 months from the placement. Approximately 40 mL of blood will be 
drawn and a urine sample may be collected for storage for future study related research at the 
repeat Tick Placement visit if the repeat xenodiagnosis procedure is more than 21 days from the 
initial tick placement.  
 
Research bloods (approximately 40 mL of blood) will be drawn at the repeat tick removal visit 
for future study related research.  
 
• The tick placement site may be the same or a different site, depending on individual 
preferences.  
• For participants repeating because of less than 14 engorged ticks recovered, the period 
between the tick placement procedures can be from the time of tick removal up to 3 
months post tick removal, and up to 1 repeat may be offered for each planned round of 
xenodiagnosis to ensure adequate collection of ticks for testing. 
 
Participants who are offered the procedure because of a previous positive xenodiagnosis do not 
need to meet entry criteria at the time of the repeat procedure. All participants must fail to meet 
any exclusion criteria at the time of tick placement.  
4.8 
Ticks 
Larval ticks will be obtained from Dr. Sam Telford from a laboratory maintained tick colony at 
Tufts Veterinary School. These ticks are hatched from eggs laid by ticks that have fed only on 
specific pathogen free laboratory animals that were purchased from established vendors. Larval 
and nymphal ticks will be maintained on CB17 SCID mice, which are highly susceptible to many 
infections since they are immunodeficient. Mice that serve as hosts to tick colony material are 
screened monthly by blood smear and PCR for the main deer tick-transmitted agents.  
 
B. burgdorferi, Babesia microti, and Anaplasma phagocytophilum (human pathogens known to 
be transmitted by I. scapularis ticks) are not transmitted transovarially (from mother to larvae). 
Deer tick virus, a flavivirus closely related to Powassan virus, has been linked to rare cases of 
human encephalitis 54,55. Deer tick virus is inherited; however, its presence is readily detected in 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
20
a laboratory colony fed upon mice because all mice die with paresis within 4-14 days. There are 
some bacteria in ticks that can be transmitted transovarially including B. miyamotoi 56,57. There 
have been reports of Bartonella spp. in Ixodes ticks, but transmission of any Bartonella spp. by 
ticks to humans has not been established 58,59.   
 
Each batch of ticks prepared for use in human xenodiagnosis will be tested for all known Ixodes 
transmitted human pathogens. Approximately one-third (⅓) of the larvae from each egg mass 
will be crushed and tested by PCR for the presence of the known human pathogens B. 
burgdorferi, B. microti, A. phagocytophilum, B. miyamotoi, Bartonella spp., deer tick virus and 
orbiviruses. In addition, a representative number of larvae from each batch (not less than 25) will 
be tested at Dr. Linden Hu’s laboratory using a panel of broad-range primers targeting a wide 
range of genera including Spirochetes (all Borrelia species), Francisella, Bartonella, Anaplasma, 
Ehrlichia, Rickettsia and Babesia. Any egg mass that tests positive for any of these organisms 
(but for the Rickettsia endosymbiont of I. scapularis, see below) will be discarded. Production of 
additional ticks will be halted until a root-cause analysis of the source of the positive samples has 
been performed and the data presented to the Medical Monitor (MM) and the site PIs.  
 
Although our ticks are tested to be free of all known human pathogens, the ticks are not sterile 
and do carry commensal organisms. Almost all I. scapularis ticks carry a rickettsial symbiont 
60,61. At this point, there is no known disease associated with this symbiont. Anecdotally, batches 
of larvae have fed on one individual 3 separate times (as many as 40 at a time). The individual 
has remained seronegative for antibodies to spotted fever group and typhus group rickettsiae, and 
has not experienced any illness associated with feeding larvae (Dr. Sam Telford, personal 
communication). Screens of mice serving as hosts for larvae, in the past, have never 
demonstrated antibody to spotted fever group rickettsiae, suggesting that the endosymbiont does 
not exit the tick by feeding. We test the xenodiagnostic ticks for Rickettsia sp. in order to have 
baseline information about their infection in case future issues arise.  
 
Recently, Tokarz et al. performed high-throughput sequencing to identify viral sequences in the 
I. scapularis genome.62 In addition to deer tick virus described above, they identified sequences 
that partially aligned with viruses of the genus Nairovirus (named South Bay virus (SBV) and 
the genus Phlebovirus (named blacklegged tick phlebovirus, BTPV). For both, the assembled 
sequences showed low similarity to other members of the genus. Moreover, they were unable to 
identify any sequences representative of potential M segments (the glycoprotein-coding 
segments). Attempts at virus isolation by inoculation of I. scapularis SBV PCR-positive pools in 
Vero, Cos7, C6/36, and 297 cell lines were unsuccessful. They also found partial sequences with 
weak homology to viruses within the order Mononegavirales, with the greatest similarity (17%) 
observed with the Nyamanini and Midway viruses.  
 
We have extended our testing panel to include additional assays for these potential viral entities 
that currently are known only from cognate nucleic acid sequences (BTPV, SBV, and deer tick 
Mononegavirales). We have screened 5 different samples of ticks (2 different lineages) that were 
used for the previous study from 2010-2013 and found that 5/5 were positive for cognate 
sequences matching SBV, 4 of 5 for BTPV, and 0/5 for deer tick mononegavirales. We conclude 
that inasmuch as these batches of ticks were used for the Phase 1 safety trial even though they 
contained cognate sequences that matched putative viral sequences, like the rickettsial 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
21
endosymbiont of I. scapularis (REIS, now known as Rickettsia buchneri)63, there is no evidence 
of ill health resulting from the feeding of these ticks on study subjects. SCID mice have 
remained uniformly healthy after feeding ticks and we do not suspect any adventitious agent in 
our tick colonies that would compromise safety, regardless of the possibility of putative agents 
that have recently been or remain to be described. We will follow this general strategy as new 
agents that are not known human pathogens are identified. If a new pathogen is identified or a 
previously non-pathogenic organism is identified as a pathogen, use of ticks in xenodiagnosis 
will be halted until we develop a test to identify the presence of that pathogen or its products.   
4.9 
Testing of the Xenodiagnostic Ticks 
Recovered larval ticks will be tested for the presence of B. burgdorferi by PCR, culture, and/or 
direct immunofluorescence. Other techniques with high sensitivity and specificity to detect B. 
burgdorferi and/or other tick-borne pathogens may be used as they become available.  
4.10 Optional Skin Biopsy 
Up to 4 optional skin biopsies will be collected to test for evidence of B. burgdorferi in the skin. 
The test results will be compared with the xenodiagnostic test results. Skin biopsies will be 
offered before the placement of ticks (either during Initial Visit or Tick Placement Visit) and 
after tick removal in patients with acute EM, untreated Lyme arthritis and PTLDS, and only after 
tick removal for participants in the Lyme disease, post-therapy group and for healthy volunteers. 
The reason for offering before and after placement for patients with acute EM, untreated Lyme 
arthritis and PTLDS is to be able to compare the results of the biopsy before and after and the 
results from the xenodiagnostic ticks, as tick feeding may increase the chance of finding B. 
burgdorferi in the skin site. Study results have shown that spirochetes were easily seen at the site 
of tick attachment after 4 days of feeding, but not on samples taken earlier 64. For participants in 
the Lyme disease, post-therapy group, recent unpublished data has shown that skin biopsies are 
negative in EM patients after 6 months of therapy. Therefore, only biopsies after tick removal 
will be done. 
4.11 Discussion of Results 
Participants will be informed that xenodiagnosis is an experimental test and therefore there are 
no guidelines to govern treatment of participants who test positive or negative by xenodiagnosis 
for the presence of B. burgdorferi. Participants will be notified of their test results by phone or in 
person at the completion of all testing of their ticks. The study doctor will discuss the 
implications of the test results with the subject.  
 
5 
RISKS 
5.1 
Xenodiagnosis 
Anticipated risks to the participants include:  
1) The development of allergic dermatitis from surgical tape used for sealing the mesh; or  
2) The development of a mild or moderately pruritic or painful local reaction to the tick bite, 
which does not require medical attention.  
 
From our experience with the 43 participants who underwent the xenodiagnosis procedure, the 
procedure is very well tolerated. All participants successfully completed the tick placement and 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
22
there were no withdrawals during the study. The most common AE was mild itching at the site, 
which was seen in 58% of participants, with a median duration of 3 days. Repeat of the 
xenodiagnosis procedure (performed in 7 participants), was similarly well tolerated with mild 
itching at the site being the most common complaint. There were no SAEs associated with the 
procedure. All these AEs resolved and did not require any intervention. No ticks escaped from 
the dressing.  
 
Problems which may also occur and be serious but are not anticipated (and are therefore 
reportable as Unanticipated Adverse Device Effects [UADEs]) include: 
 
A) the development of a skin infection (cellulitis) from scratching or skin breaks secondary to 1 
or 2 (above) or  
B) the remote possibility of developing tick paralysis 
C) the remote possibility of infection with a yet unknown pathogen 
D) the remote possibility of developing an allergy to red meat 
 
Allergic dermatitis is typically self-limited with removal of the allergen (tape). Recovery may be 
aided by the application of topical steroids.  
 
A minority of patients who have been bitten frequently by ticks will develop antibody responses 
to tick saliva and may develop a localized reaction with a granuloma. These reactions are self-
limited.  
 
Breaks in the skin caused by allergic dermatitis or scratching of tick bites can result in the 
development of cellulitis. Development of cellulitis secondary to allergic dermatitis or scratching 
is a very rare event, but could require either oral or intravenous antibiotic therapy.  
 
Although the ticks are tested for all known human diseases that are transmitted by I. scapularis, 
the ticks are not sterile and do carry commensal organisms, and there is a possibility that a new, 
unknown pathogen may be carried by the ticks. If a new pathogen is identified or a previously 
non-pathogenic organism is identified as a pathogen, use of ticks in xenodiagnosis will be halted 
until we develop a test to identify the presence of that pathogen or its products.  
 
Tick paralysis is a rare complication of tick bites. Patients present with acute symmetric 
ascending flaccid paralysis that generally begins in the lower extremities. Paralysis can ascend to 
the upper extremities, followed by cranial nerve involvement and respiratory muscle 
involvement, if the tick is not removed. Occasional patients may present with focal weakness or 
a cerebellar presentation. Removal of the tick is the definitive therapy, and patients will usually 
improve quickly after removal; however, symptoms can progress after tick removal (most 
commonly in Australian cases, associated with I. holocyclus). Most cases in North America are 
associated with the bites of Dermacentor andersoni (the wood tick) and D. variabilis (the dog 
tick), occur in children under the age of 10, particularly girls (probably because long hair make 
difficult to notice the feeding tick), and the tick is attached to the head or neck. I. holocyclus is 
the predominant species causing tick paralysis in Australia 65. I. scapularis is not associated with 
this disease, although a case associated with I. scapularis was reported in Mississippi 66. 
Assuming the tick identification was correct, it seems to be a highly unusual complication when 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
23
compared with the very high number of deer tick bites that occur yearly. A neurotoxin produced 
in the saliva of female ticks causes the paralysis. The I. holocyclus toxin has been identified and 
is named holocyclotoxin. The transcriptome analysis of over 8000 expressed sequence tags from 
6 different salivary gland cDNA libraries from I. scapularis showed only a poor match with 
holocyclotoxin, suggesting the toxin is not found in I. scapularis 67. Therefore, it is highly 
unlikely that patients in this study will develop tick paralysis as a complication of tick placement, 
as I. scapularis is not associated with this disease, ticks being used are in the larval stage (adult 
female ticks are usually the stage associated with tick paralysis), and patients in the study are 
adults. 
 
There has been an association between serious allergic reactions to red meat (including delayed 
anaphylaxis) and tick bites in some susceptible individuals 68,69. It is caused by an IgE antibody 
that binds to galactose-α-1,3-galactose (alpha-gal). Individuals with red meat allergy often 
reported a more severe local reaction to tick bites, with significant itching and redness around the 
bite. It is unknown if this allergy can develop after I. scapularis bites. The association has been 
described in Australia with I. holocyclus 68. It has also been described in patients in the southern 
US 70. A study suggested that as many as 20% of the population in Virginia, North Carolina, and 
Tennessee states, regions where Amblyomma americanum is common, have serum IgE 
antibodies to alpha-gal69. Individuals in the southeastern US (where Amblyomma is more 
prevalent) are more likely to have antibodies against alpha-gal than individuals from Boston and 
Scandinavia 69. Patients with a history of severe reactions to tick bites are excluded from 
participation in the protocol, but it is possible that exposure to ticks may trigger a future allergic 
reaction in susceptible individuals. No reports of meat allergy were seen in the Phase I trial of 
Xenodiagnosis. 
 
Anaphylaxis following a tick bite has rarely been reported 71 72-74. These reports include the 
Argus reflexus, Rhipicephalus sanguineus, I. holocyclus and I. ricinus. However, to our 
knowledge, it has not been reported in I. scapularis ticks. Recurrent anaphylaxis has been 
reported in I. pacificus, a tick that is closely related to I. scapularis 75. Allergic reactions may 
follow the bite of any of the different tick life cycle forms and is related to the release of salivary 
protein. Because these reactions are rare, it is uncertain whether repeated tick bites increase the 
risk of anaphylaxis. 
 
Repeat of the xenodiagnosis procedure may increase the risk of sensitization and local reactions. 
It is known that people can become sensitized to tick bites over time and after having had many 
tick bites. A study of I. scapularis bite reactions and their protective effect against acquisition of 
Lyme disease on Block Island, Rhode Island, showed that among 610 participants, 52% reported 
at least 1 tick bite (mean 2.2, 95% CI 2.0–2.4), and 32% reported itch with any tick bite. None 
reported anaphylaxis associated with tick bite. The probability of itch doubled as the number of 
reported tick bites increased from 1 to 2 (21% to 46%, respectively) and doubled again from 2 to 
4 reported bites (46% to 97%, respectively; linear trend p < 0.001). There was an inverse 
relationship between tick itch reaction and development of Lyme disease. The likelihood of 
Lyme disease infection decreased with >3 reports of tick-associated itch (odds ratio 0.14, 95% CI 
0.94–0.03, p = 0.01). The possible mechanisms include a heightened awareness by a person of an 
attached tick with removal of the potentially infecting tick before pathogen transmission can 
occur, premature detachment of ticks before pathogen transmission can occur, or neutralization 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
24
of some components of tick saliva that ensure successful feeding and facilitate pathogen 
transmission. 76,77 
 
There is a risk that ticks, either fed or unfed, may escape from the dressing. Loss of ticks should 
not pose a risk to either the subject or other people in contact with the subject. Ixodes ticks 
require high moisture content and would not survive in an indoor or urban setting for more than 
1 or 2 days. Unfed ticks would be uninfected and at most, would attach at a site other than the 
forearm on the subject or, possibly, on a family member. Fed ticks are not expected to feed again 
for 3-6 months. Because the ticks are tested and carry no known disease, an unfed tick that 
escaped into a suitable environment in the subject’s yard or surrounding area, would not pose a 
threat to other humans, wildlife or add a new species into the environment. Ixodes ticks are 
highly prevalent in rural and suburban Massachusetts, Rhode Island, Connecticut, New York, 
Maryland, and Virginia.  
5.2 
Blood Drawing 
The two hazards associated with blood drawing are those of venipuncture and those associated 
with blood loss. Risks of venipuncture are pain at the site of the needle stick, the potential 
formation of hematoma at the site of the needle stick, and a small potential for infection and/or 
inflammation at the site of the needle stick. Occasionally, individuals may faint as a result of 
vasovagal reactions to the procedure. The major risk of repeated blood drawing is anemia. 
Current guidelines for maximum amount of blood drawn for research will be followed.  
5.3 
Punch Skin Biopsy 
Punch biopsies have a low incidence of infection, bleeding, non-healing, or significant scarring 
78,60. Rarely, there may be injuries or damage to the structures beneath the skin site (such as an 
artery or a nerve). Patients rarely may have local allergic reactions to the tape. Severe allergic 
reactions (anaphylaxis) to lidocaine are very rare. 
 
6 
BENEFITS 
There are no direct benefits to the study participants. If the xenodiagnostic test is positive, they 
will know that they still harbor B. burgdorferi in their body. It is currently unknown whether 
treatment with more or different antibiotics will be beneficial to these patients 
 
The benefit to society is that if it is shown that patients continue to harbor bacteria after 
antibiotic therapy, this may result in a paradigm shift that will lead to new controlled therapeutic 
trials. In addition, xenodiagnosis may become a new method for diagnosing persistent infection 
after antibiotic therapy. 
 
7 
STUDY ANALYSIS 
7.1 
Sample Size Justification 
This study is powered to detect difference in the positivity rate between symptomatic and 
asymptomatic individuals. Self-reported symptoms will be characterized using symptom scales, 
the SF-36v2 questionnaire and fatigue scales. We will use a composite score derived from the 
measurement to assign symptomatic and asymptomatic status to the participants.79  
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
25
While the majority of the patients with Lyme disease who were treated with antibiotic therapy 
were negative by xenodiagnosis in our Phase 1 study, the number of patients tested in each group 
was small and the upper limit of the 95% confidence interval of each proportion is relatively high 
(up to 46%). Also, an important caveat is that the number of tested xenodiagnostic ticks per 
participant in general was also small, and the number of engorged ticks that are tested for each 
individual is an important variable, as the more engorged ticks tested, the larger the probability 
of detecting a positive. For example, if the true sensitivity for a single engorged tick to detect the 
bacteria is 1/10 (based on results of the mice studies), then one would need to recover 14 
engorged ticks to have 75% sensitivity and 22 fed ticks to have 90% sensitivity. 
 
Based in our very preliminary data, the upper limit of the exact 95% confidence interval for the 
proportion of positive xenodiagnosis in symptomatic patients (10%) was 0.445. Table 2 shows 
the sample size needed for 80% power, with a Type I error rate of 0.05, assuming varying 
assumptions for the true prevalence rates for asymptomatics (p1) and symptomatics (p2). For 
example, with a total sample size of 86 individuals, there would be 80% power to detect a 
between-group difference in the positivity rate if the true prevalence is 5% or less in the non-
symptomatics and at least 30% in the symptomatics, assuming equal numbers between the 2 
groups, (43 non-symptomatics and 43 symptomatics).  
 
Table 2: Sample Size Needed for 80% Power with a Type I Error Rate of 0.05, and Varying 
Assumptions for the True Prevalence Rates for Asymptomatics (p1) and Symptomatics 
(p2) 
 
p2 = 0.40 
p2 = 0.30 
p1= 0.05 
54 
86 
p1= 0.025 
46 
68 
p1= 0.01 
42 
60 
p1= 0 
40 
56 
 
Assuming a 10 to 20% rate of missing data due to failed screenings, assays or participant drop-
out, we plan to recruit up to 100 individuals.  
 
For the other groups post-Lyme disease syndromes (PLDS), acute EM, and untreated Lyme 
arthritis, the precision with which the rates can be estimated from the observed data depends on 
the true underlying response rate and the sample size. Exact Clopper-Pearson 2-sided 95% CIs 
for the observed positivity rate the different proposed group sizes and a range of numbers of 
positive individuals are shown in Table 3. Healthy volunteers (negative controls) results will be 
used for specificity of the testing. 
 
Table 3: Exact 95% Confidence Interval for the Observed Positivity Rate, for Varying 
Group Size (N) and Observed Numbers of Positive Individuals 
# of observed 
positives samples 
N=20 
N=40 
0 
(0.000, 0.161) 
(0.000, 0.09) 
1 
(0.009, 0.236) 
(0.004, 0.13) 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
26
2 
(0.0279, 0.301) 
(0.014, 0.165) 
5 
(0.112, 0.468) 
(0.054, 0.261) 
10 
(0.299,0.701) 
(0.142, 0.402) 
15 
(0.531, 0.888) 
(0.242, 0.529) 
20 
(0.838, 1) 
(0.352, 0.648) 
 
Data will be collected for any positive tests by xenodiagnosis. Because there is no gold standard 
test among those we will employ, comparative positivity rates between the tests will be 
determined. Summaries of patient characteristics will be assessed by study group. For categorical 
variables, the numbers and proportions will be tabulated; for continuous variables, the mean, 
median, SD, and interquartile range will be reported. Fisher’s exact text will be used to examine 
the association of 2 categorical variables. For continuous variables, t-tests and nonparametric 
methods, such as the Wilcoxon or Spearman’s correlation coefficient, may be used to assess 
associations between subgroups of patients.  
 
AEs related to xenodiagnosis will be tabulated and summaries with numbers and proportions will 
be given. 
7.2 
Anticipated Difficulties 
One anticipated difficulty is the recruitment of participants and the details of the xenodiagnostic 
protocol. In the Phase I pilot study, we did not have major issues with patient acceptance in 
either the B. burgdorferi infected or healthy control participants. One major barrier to 
recruitment in the first phase of the study was study physician availability. In the pilot phase, we 
attempted to recruit patients with EM at the time of diagnosis for immediate tick placement. We 
found that it was difficult to enroll these patients as the timing of when these participants would 
present is unpredictable and patients are understandably more concerned with their new 
diagnosis than traveling to see a study physician. We will now focus on recruiting patients in the 
interim between diagnosis and the completion of therapy, which will allow predictable 
scheduling of the participants. Also, in the past, we included private practice physicians who 
were to enroll participants. In the absence of a study infrastructure at the private practice sites, 
we found that enrollment from these sites was minimal. We are now changing our procedure so 
that the study coordinator will review the chief complaints of patients scheduled in clinic and be 
responsible for alerting the physician and then making informational calls to the patient. Patients 
who agree to enroll will be scheduled in groups and the study coordinator will travel to the 
physician’s office to assist the physician with entry and enrollment. 
 
An anticipated problem is the possibility of re-infection with B. burgdorferi after antibiotic 
therapy. Because these participants live in endemic areas with repeated exposures to ticks, it is 
possible that they may become re-infected through a new tick bite after the antibiotic treatment. 
Re-infection is a relatively uncommon event, especially in the first year after the original 
infection. Our questionnaire will specifically ask participants about occurrence of EM lesions or 
other signs of Lyme disease in the interval between antibiotic therapy and evaluation for 
enrollment in the study. It is also worth noting that, even in the unlikely event a reinfection 
occurs for one of our study participants, a positive xenodiagnostic test in this group would be an 
important finding as these would be missed by standard serological testing and these patients 
would go untreated.  

 
 
Protocol Version 10 
22 Mar 2021 
 
 
27
7.3 
Interpretation and Impact 
At the completion of this trial, we will have a better idea of what the rate of positivity for B. 
burgdorferi by xenodiagnosis is in patients with Lyme disease after treatment. We will have 
assessed whether patients with persistent symptoms of Lyme disease are more or less likely to 
have persistently detectable B. burgdorferi by xenodiagnosis. Understanding the linkage between 
persistent infection and persistent symptoms is critically important as it would affect the way that 
persistent disease is thought to develop mechanistically and would open the door to many new 
studies of both mechanism and for attempts to eradicate residual infection. 
 
8 
ASSESSMENT OF SAFETY 
8.1 
Documenting, Recording, and Reporting Adverse Events  
This trial is being conducted in compliance with 21 CFR 812 [FDA/IDE] regulations. Safety 
Reporting for this study includes additional events definitions and requirements set forth by the 
Sponsor and the IRB.  
 
At each contact with the subject, information regarding adverse events will be elicited by 
appropriate questioning and examinations and will be:  
• immediately documented in the subject’s medical record/source document, 
• recorded on the Adverse Event Case Report Form (AE CRF), and 
reported as outlined below (e.g., Sponsor, IRB, FDA). 
8.2 
Definitions 
 
The NIAID Clinical Safety Office (CSO) is responsible for sponsor safety oversight of this 
study, and the definitions below comply with CSO requirements. 
 
ADVERSE EVENT (AE) - An adverse event is any untoward or unfavorable medical 
occurrence in a human subject, including any abnormal sign (e.g., abnormal physical exam or 
laboratory finding), symptom, or disease, temporally associated with the subject’s participation 
in the research, whether or not considered related to the research. 
 
ADVERSE DEVICE EFFECT (ADE): A subset of AEs, as defined above, that are specifically 
related to the use of an investigational medical device. 
 
SERIOUS ADVERSE EVENT - an AE resulting in any of the following outcomes: 
• death 
• is life threatening (i.e., an immediate threat to life)  
• inpatient hospitalization or prolongation of an existing hospitalization 
• a persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• congenital anomaly /birth defect 
• is Medically Important* 
 
* Medical and scientific judgment should be exercised when deciding whether expedited 
reporting is appropriate in other situations, such as important medical events that may not be 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
28
immediately life threatening or result in death or result in hospitalization but may jeopardize the 
patient or may require intervention to prevent one of the other outcomes listed above. These 
should also usually be considered serious. 
 
UNEXPECTED ADVERSE EVENT - any AE (including SAEs and ADEs, as defined in this 
protocol), that occurs at a level of specificity or severity that is not consistent with the protocol. 
Expected means that the event has previously been observed with the study procedure and is 
identified and/or described in the protocol.  
 
Unanticipated Problem (UP) 
An Unanticipated Problem is any event, incident, experience, or outcome that: 
 
1.  is unexpected in terms of nature, severity, or frequency in relation to: 
a. the research risks that are described in the IRB-approved research protocol and informed 
consent document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. possibly, probably, or definitely related to participation in the research; and 
3. places participants or others at a greater risk of harm (including physical, psychological, 
economic, or social harm) than was previously known or recognized. (Per the IDE Sponsor, 
an AE with a serious outcome will be considered increased risk.) 
 
Serious Unanticipated Problem (UP): A UP that meets the definition of a Serious Adverse 
Event or compromises the safety, welfare or rights of subjects or others. 
  
Unanticipated problem that is not an Adverse Event (UPnonAE): An unanticipated problem 
that does not fit the definition of an adverse event, but which may, in the opinion of the 
investigator, involve risk to the subject, affect others in the research study, or significantly 
impact the integrity of research data. These events may involve a greater risk of social or 
economic harm to subjects or others rather than physical/psychological harm. Such events would 
be considered a non-serious UP. Examples of a UPnonAE include a breach of confidentiality, 
accidental destruction of study records, or unaccounted-for study drug. 
 
UNANTICIPATED ADVERSE DEVICE EFFECT (UADE) – “Any serious adverse effect on 
health or safety or any life-threatening problem or death caused by, or associated with, a device, 
if that effect, problem, or death was not previously identified in nature, severity, or degree of 
incidence in the investigational plan or application (including a supplementary plan or 
application), or any other unanticipated serious problem associated with a device that relates to 
the rights, safety, or welfare of subjects.” (21 CFR 812.3) In summary, UADEs are serious, 
unanticipated, and related (possibly, probably or definitely) to the device.  
 
Anticipated AEs related to the study device (xenodiagnosis) include the following: 
• allergic dermatitis from surgical tape,  
• mild or moderate pruritic local reactions and mild or moderate painful local reactions to 
the tick bite that do not require medical intervention.  
 
All AEs and ADEs that are identified from the time the xenodiagnostic ticks are placed through 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
29
30 days after the last use of the device (ticks) will be documented in the subject’s medical record 
and will be recorded in the study database. 
 
NOTE: AEs and ADEs of any severity could be considered serious (e.g., medically important) 
based on the medical judgment of the investigator. For example, cellulitis of any severity grade 
should be considered serious. 
 
Protocol Deviation: Any change, divergence, or departure from the IRB-approved research 
protocol.  
1. Major Deviation – Deviation from the IRB-approved protocol that have, or may have the 
potential to, negatively impact, the rights, welfare or safety of the subject, or to 
substantially negatively impact the scientific integrity or validity of the study. 
2. Minor Deviation – A Deviation that does not have the potential to negatively impact the 
rights, safety, or welfare of subjects or others, or the scientific integrity or validity of the 
study. 
 
Non-compliance: Failure of an investigator to follow the applicable laws, regulations, or 
institutional policies governing the protection of human subjects in research, or the requirements 
or determinations of the IRB, whether the failure is intentional or not. 
• Continuing Non-compliance – A pattern of recurring non-compliance that either has 
resulted, or, if continued, may result in harm to subjects or otherwise materially 
compromise the rights, welfare and/or safety of subjects, affect the scientific integrity of 
the study or validity of the results. The pattern may comprise repetition of the same non-
compliant action(s), or different non-compliant events. Such non-compliance may be 
unintentional (e.g. due to lack of understanding, knowledge, or commitment), or 
intentional (e.g. due to deliberate choice to ignore or compromise the requirements of any 
applicable regulation, organizational policy, or determination of the IRB). 
• Serious Non-compliance – Non-compliance, whether intentional or not, that results in 
harm or otherwise materially compromises the rights, welfare and/or safety of the subject. 
Non-compliance that materially affects the scientific integrity or validity of the research 
may be considered serious non-compliance, even if it does not result in direct harm to 
research subjects. 
8.3 
Investigator Evaluation Adverse Events and Adverse Device Effects 
The Investigator will evaluate all potential AEs and ADEs with respect to:  
 
Seriousness (criteria listed in Section 8.2. above),  
 
Anticipated vs. Unanticipated (for devices as defined in Section 8.2 above), 
 
Expected vs. Unexpected (for study procedures as defined in Section 8.2 above),  
 
Severity (intensity or grade – Section 8.3.1 below), 
 
Causality (relationship to the study device or to a study procedure) according to the  
 
 
 guidelines in Section 8.3.2 
8.3.1 Severity 
Grading of AEs will conform to the toxicity scale in Appendix 3. For items not described in 
Appendix 3, the Division of AIDS Table for Grading Adult and Pediatric Adverse Experiences 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
30
Version 1.0 - December 2004 (Clarification dated August 2009) will be used (https://rsc.tech-
res.com/docs/default-
source/safety/table_for_grading_severity_of_adult_pediatric_adverse_events.pdf?sfvrsn=6).  
 
All clinical AEs that are not listed in the toxicity scale will be assessed for severity and classified 
into one the categories below: 
 
• Grade 1 (Mild): Symptoms causing no or minimal interference with usual social & 
functional activities or represent mild increase over baseline 
• Grade 2 (Moderate): Symptoms causing greater than minimal interference with usual 
social & functional activities or represent moderate increase over baseline  
• Grade 3 (Severe): Symptoms causing inability to perform usual social & functional 
activities or represent severe increase over baseline.  
• Grade 4 (Life threatening): Symptoms causing inability to perform basic self-care 
functions OR Medical or operative intervention indicated to prevent permanent 
impairment, persistent disability, or death  
• Grade 5 (Death) 
 
8.3.2 
Causality 
All AEs will be assessed by the investigator for relationship to study procedures and the study 
device. A causal relationship (possible, probable, or definite) means that a research procedure or 
the study device caused or is reasonably likely to have caused the AE.  
 
The best estimate of the causal relationship between the study device and an AE will be made 
by the investigator using the following causality categories:  
 
Definitely Related 
• reasonable temporal relationship 
• follows a known response pattern 
• clear evidence to suggest a causal relationship 
• there is no alternative etiology 
 
Probably Related 
 
• reasonable temporal relationship 
• follows a suspected response pattern for the procedure or the device  
• no evidence of a more likely alternative etiology 
 
Possibly Related 
• reasonable temporal relationship 
• little evidence for a more likely alternative etiology 
 
Unlikely Related 
 
• does not have a reasonable temporal relationship OR 
• good evidence for a more likely alternative etiology 
 
 
Not Related 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
31
• does not have a temporal relationship OR 
• definitely due to an alternative etiology  
8.4 
Reporting Serious Adverse Events, Unanticipated Adverse Device Effects, and 
Unanticipated Problems to the IDE Sponsor 
8.4.1 Adverse Events 
Line listings, frequency tables, and other summary AE data will be submitted to the Sponsor 
when needed for periodic safety assessments, review of IDE annual reports, review of IDE safety 
reports, and preparation of final study reports. 
8.4.2 Serious Adverse Events 
All SAEs (regardless of relationship and whether or not they are also UPs) must be reported on the 
Safety Expedited Report Form (SERF) and sent to the Sponsor CSO by fax or e-mail attachment. 
Deaths and immediately life threatening SAEs must be reported to the CSO within 1 business 
day after the site becomes aware of the event. All other SAEs must be reported within 3 business 
days of site awareness.  
 
SPONSOR CLINICAL SAFETY OFFICE CONTACT INFORMATION:  
 
Clinical Safety Office 
5705 Industry Lane 
Frederick, MD 21704 
Phone 301-846-5301 
Fax 301-846-6224 
E-mail: rchspsafety@mail.nih.gov  
8.4.3 Unanticipated Problems 
Unanticipated problems that are also AEs must be reported to the CSO and sent by fax or e-mail 
attachment no later than 7 calendar days of site awareness of the event. UPnonAEs are not 
reported to the Sponsor CSO. 
 
Report all UPs that are also AEs to the CSO on the NIH Problem Report Form. 
8.4.4 Unanticipated Adverse Device Effects (UADEs) 
All ADEs that are identified from the time the xenodiagnostic ticks are placed through 30 days 
after the last use of the device will be documented in the subject’s medical record and will be 
recorded in the study database.  
 
UADEs (unanticipated, serious, and possibly, probably, or definitely related to the device) must 
be recorded on a SERF, clearly identified as a UADE, and sent to the CSO (addressed in Section 
8.3.1) within 10 working days. 
 
21CFR812.46 requires the Sponsor to conduct an investigation of all UADE reports. The results 
of the investigation will be reported to the FDA, all reviewing IRBs, and all participating 
investigators within 10 working days after the Sponsor becomes aware of the UADE. 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
32
 
If the Sponsor determines that a UADE presents an unreasonable risk to participants, the Sponsor 
shall terminate all investigations, or parts of investigations, presenting that risk as soon as 
possible but no later than 5 working days after the Sponsor makes this determination and no later 
than 15 working days after the Sponsor first received notice of the effect. The investigation may 
not resume without subsequent IRB and FDA approval (21CFR812.150) 
8.5 
Follow-up of Adverse Events and Adverse Device Effects 
AEs temporally associated with study procedures and ADEs that occur from the time the 
informed consent is signed are followed until final outcome is known. AEs temporally associated 
with a study procedure that have not resolved by the end of the study follow-up period are 
recorded on the AE CRF as ONGOING. 
 
ADEs that have not resolved by the end of the study follow-up period are followed until final 
outcome is known. If it is not possible to obtain a final outcome for an ADE (e.g., the subject 
is lost to follow-up), the reason a final outcome could not be obtained will be documented by 
the investigator. 
8.6 
Pausing Rules for the Protocol 
The PI will closely monitor and analyze study data as it becomes available and will make 
determinations regarding the occurrence and grading of AEs. AEs will be evaluated with regard 
to the known complications associated with the study procedure (xenodiagnosis) and the 
presence of concurrent medications or concurrent illnesses. Upon determination of any Grade 4 
AE or any 2 Grade 3 AEs (in different participants) thought to be definitely, probably, or 
possibly related to the study procedure, the study will be halted (no new enrollments and no 
further use of the study procedure by the investigators) and a UADE report will be submitted to 
the local IRB and to the OCRPRO Safety Office. Approval by each IRB and the FDA is required 
before the study can resume.  
 
The IRBs, the NIAID, the FDA, or other government agencies, as part of their duties to ensure 
that research participants are protected may discontinue the study at any time. Subsequent review 
of UADEs by the IRB, the Sponsor, the FDA, and other regulatory authorities may also result in 
suspension of further trial interventions/administration of the study procedure at a site. The FDA, 
other regulatory authorities, and the Sponsor retain the authority to suspend additional 
enrollment and Study Agent/Intervention administration for the entire study as applicable.  
 
9 
DATA AND SAFETY MONITORING PLAN 
 
The PI will monitor protocol data (through regular communication with the study teams at the 
other sites) that could affect subject safety or confidentiality by communicating at pre-arranged 
intervals with the Site PIs. Information to be shared includes AEs, documentation of subject 
evaluation, treatment and disposition, and coding of subject samples and data. Serious and other 
AEs will be reported to the site’s IRB and the CSO as outlined above.  
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
33
9.1.1 Safety Review and Communications Plan 
A Safety Review and Communications Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the PI and the CSO, which delineates 
the safety oversight responsibilities of the PI, the CSO, and other stakeholders. The SRCP also 
includes the overall plan for conducting periodic safety surveillance assessments. 
 
9.1.2  Sponsor Medical Monitor 
A SMM, representing the Sponsor (OCRPRO), has been appointed for oversight of safety in this 
clinical study. The SMM will be responsible for performing safety assessments as outlined in an 
SRCP. 
 
A monthly (or other specified frequency) conference call with the SMM, the PI, and members of 
the study teams from all sites will review all AE safety data (Grade 1 or higher) as specified in 
the SRCP. A safety report will be prepared for distribution to the safety team, and will include 
data elements specified in the SRCP. 
 
10 REPORTING PROCEDURES 
10.1 Investigator Reporting Responsibilities to the IRB 
Each site will report AEs to their local IRB according to their IRB’s policy.  
10.1.1 Reporting Procedures at the NIH 
Reportable events will be tracked and submitted to the NIH IRB as outlined in Policy 801. 
10.1.2 Investigator Reporting Responsibilities to Local IRB(s)  
Investigators are responsible for submitting UADE Reports and UP summaries that are received 
from the Sponsor to their local IRB/Ethics Committee. Investigators must also comply with all 
local IRB/Ethics Committee reporting requirements.  
10.2 Reporting to the NIAID Clinical Director 
At the NIH site, the PI will report UPs, major protocol deviations, and deaths to the NIAID 
clinical director according to institutional timelines. 
 
11 FINANCIAL COMPENSATION 
Participants will be paid the established NIH daily rate for participation in particular aspects of 
the protocol. As of 2015, outpatient visits rates are $20 for the first hour and $10 for each 
additional hour. Separate additional payments will be provided for the following: 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
34
Table 4: Financial Compensation 
TEST 
PAYMENT 
Skin Biopsies 
$25 for each skin biopsy 
Xenodiagnosis 
$100 
 
At the NIH site, payment will be provided through the NIH Clinical Center Payment Office as a 
check, direct deposit, or automated clearing house payment after the completion of each study 
visit. Payment or reimbursement for travel-related expenses (e.g., transportation, hotel, meals) 
will be provided according to the NIAID Travel Policy. 
 
12 PLAN FOR MAINTAINING THE PRIVACY AND CONFIDENTIALITY OF 
SUBJECT RECORDS 
 
Subject records will be kept at Medical Records Center for each site. Research records will be 
kept in a locked file cabinet, and only the site PI or associate investigators will have access to the 
research records, unless required by law. The investigator (and/or designee) will make study 
documents (including source medical records) readily available for inspection by the local IRB, 
the FDA, the site monitors, and the Sponsor for confirmation of the study data. 
 
13 INTENDED USE OF THE SAMPLES/SPECIMENS/DATA 
 
Blood, urine, and skin samples collected under this study may be used to test for known and yet 
unknown pathogens, immunological and inflammatory markers, and host reactivity to tick 
salivary protein. Patients may choose not to have their specimens stored or used for research on 
unrelated diseases and conditions.  
 
Samples and data collected under this protocol will be used to study Lyme disease and related 
disorders. No human genetic testing will be done on these samples.  
 
How samples/specimen/data will be stored 
Access to research samples will be limited using either a locked room or a locked freezer. 
Samples and data will be stored using codes assigned by the investigators or their designee(s). 
Data will be kept in password-protected computers. The key linking the study code to identifiers 
will be maintained at each site and will only be accessible to investigators or designees at that 
site.  
 
How samples/specimen/data will be tracked 
Samples will be tracked using Excel or BSI databases. Samples may also be stored and tracked 
utilizing the Frederick National Laboratory for Cancer Research (FNLCR) repository operated 
by Leidos Biomedical Research, Inc.  
 
What will happen to the samples/specimen/data at the completion of the protocol?  
In the future, other investigators (both at NIH and outside) may wish to use these samples and/or 
data for research purposes. If the planned research falls within the category of “human subjects 
research” on the part of the NIH researchers, NIH IRB review and approval will be obtained. 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
35
This includes the NIH researchers sending out coded and linked samples or data and getting 
results that they can link back to their subjects..  
 
What circumstances would prompt the PI to report to the IRB loss or destruction of 
samples/specimen/data? 
Any loss or unanticipated destruction of samples or data (for example, due to freezer 
malfunction) that meets the definition of a reportable event will be reported to the NIH IRB 
according to NIH HRPP Policy 801. 
 
Any loss or unanticipated destruction of more than 30% of samples or data that compromises the 
scientific integrity of the study will be reported to the IRB. 
 
14 DATA MANAGEMENT PLAN 
Study data will be collected at the study site(s) and maintained on standardized electronic or 
paper case report forms (CRFs), or an electronic data system. Research data may also be kept 
using Excel (with appropriate controls to protect data integrity and subject confidentiality) as 
well as multiple programs for statistical analysis. 
 
These forms or systems are to be completed on an ongoing basis during the study. The CRFs and 
instructions will be distributed to the site(s) by the Sponsor or printed by the sites themselves 
after Sponsor approval. Data entries on paper CRFs must be completed legibly with black 
ballpoint pen. Corrections must be made by striking through the incorrect entry with a single line 
(taking care not to obliterate or render the original entry illegible) and entering the correct 
information adjacent to the incorrect entry. Corrections to paper CRFs must be initialed and 
dated by the person making the correction. Data entered into electronic CRFs or data systems 
shall be performed by authorized individuals. Corrections to electronic CRFs or data systems 
shall be tracked electronically (password protected) with time, date, individual making the 
correction, and what was changed.  
 
The Investigator is responsible for assuring that the data collected is complete, accurate, and 
recorded in a timely manner. Source documentation (the point of initial recording of information) 
should support the data collected on the CRF, and must be signed and dated by the person 
recording and/or reviewing the data. All CRFs should be reviewed by the Investigator and signed 
as required with written or electronic signature, as appropriate. Source documents include all 
recordings of observations or notations of clinical activities, and all reports and records necessary 
for the evaluation and reconstruction of the clinical trial. Source documents include, but are not 
limited to, the subject’s medical records, laboratory reports, ECG tracings, x-rays, radiologist’s 
reports, subject’s diaries, biopsy reports, ultrasound photographs, progress notes, pharmacy 
records, and any other similar reports or records of procedures performed during the subject’s 
participation in the study. Data from CRFs, CRIMSON Data System, or other electronic database 
will be collected directly from participants during study visits and telephone calls, or will be 
abstracted from participants’ diaries and medical records. The subject’s medical record must 
record his/her participation in the clinical trial and, what medications (with doses and frequency) 
or other medical interventions or treatments were administered, as well as any adverse reactions 
experienced during the trial.  
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
36
Study results: Each subject will receive a letter at the completion of the study to discuss the 
findings of the study. The letter will be prepared and submitted to the IRB for approval before 
distribution. 
 
15 SUBJECT RECRUITMENT 
 
Lyme disease affects persons in all age groups, with peaks occurring for children aged 5-9 years 
and adults 55-59 years. Males and females are nearly equally affected in all age groups. The 
racial breakdown of persons with Lyme disease, according to the national surveillance data from 
1992 to 2006 [54], shows a preponderance of white (94.1%). This reflects the demographic 
profile of the population at risk, both by geographic location and behavioral risk factors. Current 
scientific evidence does not indicate that the natural history of Lyme disease differs based on 
gender or racial minority status. All efforts will be made to recruit patients whose gender or 
minority represents the general population. Only adult participants will be recruited for this 
study. Patients may self-refer or be referred by a physician to the protocol. At this point, no 
advertisement is planned, but may be used in the future. If that is the case, it will be submitted to 
each site’s IRB for approval. 
 
Participants will be recruited at 5 study sites, 3 of which (NIH, Tufts and Mansfield) participated 
in the Phase 1 xenodiagnostic study. The current study sites include the NIH Clinical Center, 
Tufts Medical Center, Mansfield Family Practice, Stony Brook University (State University of 
New York), and NYMC.  
 
The NIAID Lyme disease program at the NIH Clinical Center has evaluated more than 400 
patients. For this study, we have established collaboration with Dr. John Aucott at Johns Hopkins 
University, who has a large referral network for a longitudinal study of patients with EM that has 
already enrolled over 100 participants. Dr. Aucott will be referring patients to the NIH site. 
 
Tufts Medical Center is a tertiary referral center for Lyme disease seeing over 100 cases per 
year. The majority of these cases are patients experiencing persistent symptoms including 
arthritis. Tufts will also oversee recruitment of participants by Dr. Kenneth Dardick, who directs 
a large Family Medicine Practice in Mansfield, Connecticut. Dr. Dardick has been involved in 
Lyme disease research for more than 20 years. His clinic cares for about 100 patients with acute 
Lyme disease and 25 patients with persistent symptoms per year.  
  
Dr. Wormser has enrolled over 1200 patients with Lyme disease in various studies on 
pathogenesis, diagnosis, clinical manifestations and treatment. Lyme disease patients were 
mostly recruited from the walk-in Lyme Disease Diagnostic Center at New York Medical 
College, now in its 25th year of existence.  
 
Dr. Marcos is an infectious diseases physician at Stony Brook University Department of 
Medicine. He is the current director for the outpatient clinic for tick borne diseases overseeing 
more than 400 patients with any tick borne disease in Suffolk County.  
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
37
16 CONSENT PROCEDURES 
 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation. Extensive discussion of 
risks and possible benefits of this therapy will be provided to the participants and their families. 
Consent forms describing in detail the study interventions/products, study procedures, and risks 
are given to the subject and written documentation of informed consent is required prior to 
starting intervention/administering study product. Consent forms will be IRB-approved and the 
subject will be given the opportunity to read and review the document. Upon reviewing the 
document, the investigator or designee will explain the research study to the subject and answer 
any questions that may arise. The participants will sign the informed consent document prior to 
any procedures being done specifically for the study. The participants should have the 
opportunity to discuss the study with their surrogates or think about it prior to agreeing to 
participate. The participants may withdraw consent at any time throughout the course of the trial. 
A copy of the informed consent document will be given to the participants for their records.  
 
Consent for NIH staff will be obtained as detailed above with following additional protections: 
Consent from staff members will be obtained by an individual independent of the staff member’s 
team whenever possible.  Otherwise, the consent procedure will be independently monitored by 
the CC Department of Bioethics Consultation Service in order to minimize the risk of undue 
pressure on the staff member. 
 
17 STUDY RECORDS RETENTION 
 
The investigator is responsible for retaining all essential documents listed in the ICH GCP 
Guideline. All essential documentation for all study participants is to be maintained by the 
investigators in a secure storage facility for a minimum of 3 years. The FDA requires study 
records to be retained for up to 2 years after marketing approval or disapproval (21 CFR 
812.140), or until at least 2 years have elapsed since the formal discontinuation (terminated or 
completed) of clinical development of the investigational agent for a specific indication. These 
records are also to be maintained in compliance with IRB/EC, state, and federal medical records 
retention requirements, whichever is longest. All stored records are to be kept confidential to the 
extent provided by federal, state, and local law. It is the investigator’s responsibility to retain 
copies of source documents until receipt of written notification to the contrary from the 
OCRPRO of the NIAID. No study document should be destroyed without prior written 
agreement between OCRPRO/NIAID and the PIs. Should the investigator wish to assign the 
study records to another party and/or move them to another location, the investigator must 
provide written notification of such intent to OCRPRO/NIAID with the name of the person who 
will accept responsibility for the transferred records and/or their new location. NIAID must be 
notified in writing and written NIAID permission must be received by the site prior to 
destruction or relocation of research records. 
 
18 PROTOCOL MONITORING 
 
As per ICH-GCP 5.18 and FDA 21 CFR 812.43(d) clinical protocols are required to be 
adequately monitored by the study Sponsor. This study monitoring will be conducted according 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
38
to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under contract to the 
NIAID/OCRPRO will visit the clinical research sites to monitor all aspects of the study in 
accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent documents for 
each monitored subject; 2) to verify the prompt and accurate recording of all monitored data 
points and prompt reporting of all SAEs; 3) to compare abstracted information with individual 
participants’ records and source documents (participants’ charts, laboratory analyses and test 
results, physicians’ progress notes, nurses’ notes, and any other relevant original subject 
information); and 4) to ensure protection of study participants, investigators’ compliance with 
the protocol, and completeness and accuracy of study records. The monitors also will inspect the 
clinical site regulatory files to ensure that regulatory requirements (Office for Human Research 
Protections [OHRP] as applicable) and applicable guidelines (ICH-GCP and FDA 21 CFR 812) 
are being followed. During the monitoring visits, the investigator (and/or designee) and other 
study personnel will be available to discuss the study progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms, and pertinent 
hospital or clinical records) readily available for inspection by the local IRB, the site monitors, 
and the NIAID staff for confirmation of the study data.  
 
A specific protocol monitoring plan will be discussed with the PI and study staff prior to 
enrollment. The plan will outline the frequency of monitoring visits based on such factors as 
study enrollment, data collection status and regulatory obligations. 
 
19 CERTIFICATE OF CONFIDENTIALITY 
To further protect the privacy of study participants, a Certificate of Confidentiality has been 
issued by the NIH. This certificate protects identifiable research information from forced 
disclosure. It allows the investigator and others who have access to research records to refuse to 
disclose identifying information on research participation in any civil, criminal, administrative, 
legislative, or other proceeding, whether at the federal, state, or local level. By protecting 
researchers and institutions from being compelled to disclose information that would identify 
research participants, Certificates of Confidentiality help achieve the research objectives and 
promote participation in studies by helping assure confidentiality and privacy to participants. 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
39
REFERENCES 
 
1 
Bacon, R. M., Kugeler, K. J., Mead, P. S., Centers for Disease, C. & Prevention. 
Surveillance for Lyme disease--United States, 1992-2006. MMWR Surveill Summ 57, 1-9 
(2008). 
2 
Smith, R. P. et al. Clinical characteristics and treatment outcome of early Lyme disease in 
patients with microbiologically confirmed erythema migrans. Ann Intern Med 136, 421-
428 (2002). 
3 
Nowakowski, J. et al. Long-term follow-up of patients with culture-confirmed Lyme 
disease. Am J Med 115, 91-96 (2003). 
4 
Wormser, G. P. et al. Duration of antibiotic therapy for early Lyme disease. A 
randomized, double-blind, placebo-controlled trial. Ann Intern Med 138, 697-704 (2003). 
5 
Wormser, G. P. et al. The clinical assessment, treatment, and prevention of lyme disease, 
human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the 
Infectious Diseases Society of America. Clin Infect Dis 43, 1089-1134, 
doi:10.1086/508667 (2006). 
6 
Dattwyler, R. J., Halperin, J. J., Volkman, D. J. & Luft, B. J. Treatment of late Lyme 
borreliosis--randomised comparison of ceftriaxone and penicillin. Lancet 1, 1191-1194 
(1988). 
7 
Pfister, H. W. et al. Randomized comparison of ceftriaxone and cefotaxime in Lyme 
neuroborreliosis. J Infect Dis 163, 311-318 (1991). 
8 
Steere, A. C. et al. Treatment of Lyme arthritis. Arthritis Rheum 37, 878-888 (1994). 
9 
Kalish, R. A. et al. Evaluation of study patients with Lyme disease, 10-20-year follow-
up. Journal of Infectious Diseases 183, 453-460 (2001). 
10 
Kindstrand, E., Nilsson, B. Y., Hovmark, A., Pirskanen, R. & Asbrink, E. Peripheral 
neuropathy in acrodermatitis chronica atrophicans - effect of treatment. Acta Neurol 
Scand 106, 253-257 (2002). 
11 
Berglund, J., Stjernberg, L., Ornstein, K., Tykesson-Joelsson, K. & Walter, H. 5-y 
Follow-up study of patients with neuroborreliosis. Scand J Infect Dis 34, 421-425, 
doi:10.1080/00365540110080421 (2002). 
12 
Dattwyler, R. J. et al. A comparison of two treatment regimens of ceftriaxone in late 
Lyme disease. Wien Klin Wochenschr 117, 393-397 (2005). 
13 
Borg, R. et al. Intravenous ceftriaxone compared with oral doxycycline for the treatment 
of Lyme neuroborreliosis. Scand J Infect Dis 37, 449-454, 
doi:10.1080/00365540510027228 (2005). 
14 
Oksi, J. et al. Duration of antibiotic treatment in disseminated Lyme borreliosis: a 
double-blind, randomized, placebo-controlled, multicenter clinical study. Eur J Clin 
Microbiol Infect Dis 26, 571-581, doi:10.1007/s10096-007-0340-2 (2007). 
15 
Weber, K. et al. Antibiotic therapy of early European Lyme borreliosis and 
acrodermatitis chronica atrophicans. Ann N Y Acad Sci 539, 324-345 (1988). 
16 
Strle, F. et al. Azithromycin versus doxycycline for treatment of erythema migrans: 
clinical and microbiological findings. Infection 21, 83-88 (1993). 
17 
Dattwyler, R. J. et al. Ceftriaxone compared with doxycycline for the treatment of acute 
disseminated Lyme disease. N Engl J Med 337, 289-294 (1997). 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
40
18 
Picha, D., Moravcova, L., Lasikova, S., Holeckova, D. & Maresova, V. Symptoms of 
post-Lyme syndrome in long-term outcome of patients with neuroborreliosis. Scand J 
Infect Dis 38, 747-748 (2006). 
19 
Aucott, J. N., Rebman, A. W., Crowder, L. A. & Kortte, K. B. Post-treatment Lyme 
disease syndrome symptomatology and the impact on life functioning: is there something 
here? Qual Life Res 22, 75-84, doi:10.1007/s11136-012-0126-6 (2013). 
20 
Steere, A. C. et al. Treatment of the early manifestations of Lyme disease. Ann Intern 
Med 99, 22-26 (1983). 
21 
Dattwyler, R. J., Volkman, D. J., Conaty, S. M., Platkin, S. P. & Luft, B. J. Amoxycillin 
plus probenecid versus doxycycline for treatment of erythema migrans borreliosis. Lancet 
336, 1404-1406 (1990). 
22 
Weber, K. et al. A randomized trial of ceftriaxone versus oral penicillin for the treatment 
of early European Lyme borreliosis. Infection 18, 91-96 (1990). 
23 
Shadick, N. A. et al. The long-term clinical outcomes of Lyme disease. A population-
based retrospective cohort study. Ann Intern Med 121, 560-567 (1994). 
24 
Asch, E. S., Bujak, D. I., Weiss, M., Peterson, M. G. & Weinstein, A. Lyme disease: an 
infectious and postinfectious syndrome. J Rheumatol 21, 454-461 (1994). 
25 
Hunfeld, K. P., Ruzic-Sabljic, E., Norris, D. E., Kraiczy, P. & Strle, F. In vitro 
susceptibility testing of Borrelia burgdorferi sensu lato isolates cultured from patients 
with erythema migrans before and after antimicrobial chemotherapy. Antimicrob Agents 
Chemother 49, 1294-1301 (2005). 
26 
Klempner, M. S. et al. Two controlled trials of antibiotic treatment in patients with 
persistent symptoms and a history of Lyme disease. N Engl J Med 345, 85-92, 
doi:10.1056/NEJM200107123450202 (2001). 
27 
Fallon, B. A. et al. A randomized, placebo-controlled trial of repeated IV antibiotic 
therapy for Lyme encephalopathy. Neurology (2007). 
28 
Aguero-Rosenfeld, M. E., Wang, G., Schwartz, I. & Wormser, G. P. Diagnosis of lyme 
borreliosis. Clin Microbiol Rev 18, 484-509, doi:10.1128/CMR.18.3.484-509.2005 
(2005). 
29 
Wilske, B., Fingerle, V. & Schulte-Spechtel, U. Microbiological and serological 
diagnosis of Lyme borreliosis. FEMS Immunol Med Microbiol 49, 13-21 (2007). 
30 
Fleming, R. V. et al. Pre-treatment and post-treatment assessment of the C(6) test in 
patients with persistent symptoms and a history of Lyme borreliosis. Eur J Clin 
Microbiol Infect Dis (2004). 
31 
Marques, A. R., Hornung, R. L., Dally, L. & Philipp, M. T. Detection of immune 
complexes is not independent of detection of antibodies in Lyme disease patients and 
does not confirm active infection with Borrelia burgdorferi. Clin Diagn Lab Immunol 12, 
1036-1040, doi:10.1128/CDLI.12.9.1036-1040.2005 (2005). 
32 
Krupp, L. B. et al. Study and treatment of post Lyme disease (STOP-LD): a randomized 
double masked clinical trial. Neurology 60, 1923-1930 (2003). 
33 
IDSA. From the President: IDSA Stands Up For Lyme Disease Guidelines., < 
http://www.idsociety.org/newsArticle.aspx?id=4262> (2007). 
34 
Bockenstedt, L. K., Mao, J., Hodzic, E., Barthold, S. W. & Fish, D. Detection of 
attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after 
antibiotic treatment. J Infect Dis 186, 1430-1437, doi:10.1086/345284 (2002). 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
41
35 
Straubinger, R. K., Summers, B. A., Chang, Y. F. & Appel, M. J. Persistence of Borrelia 
burgdorferi in experimentally infected dogs after antibiotic treatment. J Clin Microbiol 
35, 111-116 (1997). 
36 
Straubinger, R. K., Straubinger, A. F., Summers, B. A. & Jacobson, R. H. Status of 
Borrelia burgdorferi infection after antibiotic treatment and the effects of corticosteroids: 
An experimental study. J Infect Dis 181, 1069-1081, doi:10.1086/315340 (2000). 
37 
Yrjanainen, H. et al. Anti-tumor necrosis factor-alpha treatment activates Borrelia 
burgdorferi spirochetes 4 weeks after ceftriaxone treatment in C3H/He mice. J Infect Dis 
195, 1489-1496, doi:10.1086/513873 (2007). 
38 
Hodzic, E., Feng, S., Holden, K., Freet, K. J. & Barthold, S. W. Persistence of Borrelia 
burgdorferi following antibiotic treatment in mice. Antimicrob Agents Chemother 52, 
1728-1736, doi:10.1128/AAC.01050-07 (2008). 
39 
Embers, M. E. et al. Persistence of Borrelia burgdorferi in rhesus macaques following 
antibiotic treatment of disseminated infection. PLoS One 7, e29914, 
doi:10.1371/journal.pone.0029914 (2012). 
40 
Barthold, S. W. et al. Ineffectiveness of tigecycline against persistent Borrelia 
burgdorferi. Antimicrob Agents Chemother 54, 643-651, doi:10.1128/AAC.00788-09 
(2010). 
41 
Liveris, D. et al. Comparison of five diagnostic modalities for direct detection of Borrelia 
burgdorferi in patients with early Lyme disease. Diagn Microbiol Infect Dis 73, 243-245, 
doi:10.1016/j.diagmicrobio.2012.03.026 (2012). 
42 
Liveris, D. et al. Quantitative detection of Borrelia burgdorferi in 2-millimeter skin 
samples of erythema migrans lesions: correlation of results with clinical and laboratory 
findings. J Clin Microbiol 40, 1249-1253 (2002). 
43 
Nowakowski, J. et al. Laboratory diagnostic techniques for patients with early Lyme 
disease associated with erythema migrans: a comparison of different techniques. Clin 
Infect Dis 33, 2023-2027, doi:10.1086/324490 (2001). 
44 
Schwartz, I. et al. Diagnosis of early Lyme disease by polymerase chain reaction 
amplification and culture of skin biopsies from erythema migrans lesions. J Clin 
Microbiol 30, 3082-3088 (1992). 
45 
Iyer, R. et al. Detection of Borrelia burgdorferi nucleic acids after antibiotic treatment 
does not confirm viability. J Clin Microbiol 51, 857-862, doi:10.1128/JCM.02785-12 
(2013). 
46 
Hodzic, E., Imai, D., Feng, S. & Barthold, S. W. Resurgence of persisting non-cultivable 
Borrelia burgdorferi following antibiotic treatment in mice. PLoS One 9, e86907, 
doi:10.1371/journal.pone.0086907 (2014). 
47 
Bockenstedt, L. K., Gonzalez, D. G., Haberman, A. M. & Belperron, A. A. Spirochete 
antigens persist near cartilage after murine Lyme borreliosis therapy. J Clin Invest 122, 
2652-2660, doi:10.1172/JCI58813 (2012). 
48 
Shih, C. M., Chao, L. L. & Yu, C. P. Chemotactic migration of the Lyme disease 
spirochete (Borrelia burgdorferi) to salivary gland extracts of vector ticks. Am J Trop 
Med Hyg 66, 616-621 (2002). 
49 
Marques, A. et al. Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-
human study. Clin Infect Dis 58, 937-945, doi:10.1093/cid/cit939 (2014). 
50 
Hu, L. Lyme arthritis. Infect Dis Clin North Am 19, 947-961, 
doi:10.1016/j.idc.2005.07.007 (2005). 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
42
51 
Steere, A. C. et al. Successful parenteral penicillin therapy of established Lyme arthritis. 
N Engl J Med 312, 869-874 (1985). 
52 
Nocton, J. J. et al. Detection of Borrelia burgdorferi DNA by polymerase chain reaction 
in synovial fluid from patients with Lyme arthritis. N Engl J Med 330, 229-234, 
doi:10.1056/NEJM199401273300401 (1994). 
53 
Steere, A. C., Schoen, R. T. & Taylor, E. The clinical evolution of Lyme arthritis. Ann 
Intern Med 107, 725-731 (1987). 
54 
El Khoury, M. Y. et al. Diagnosis of acute deer tick virus encephalitis. Clin Infect Dis 56, 
e40-47, doi:10.1093/cid/cis938 (2013). 
55 
Hinten, S. R. et al. Increased recognition of Powassan encephalitis in the United States, 
1999-2005. Vector Borne Zoonotic Dis 8, 733-740 (2008). 
56 
Krause, P. J. et al. Human Borrelia miyamotoi infection in the United States. N Engl J 
Med 368, 291-293, doi:10.1056/NEJMc1215469 (2013). 
57 
Gugliotta, J. L., Goethert, H. K., Berardi, V. P. & Telford, S. R., 3rd. 
Meningoencephalitis from Borrelia miyamotoi in an immunocompromised patient. N 
Engl J Med 368, 240-245, doi:10.1056/NEJMoa1209039 (2013). 
58 
Telford, S. R., 3rd & Wormser, G. P. Bartonella spp. transmission by ticks not 
established. Emerg Infect Dis 16, 379-384, doi:10.3201/eid1603.090443 (2010). 
59 
Reis, C. et al. Vector competence of the tick Ixodes ricinus for transmission of Bartonella 
birtlesii. PLoS Negl Trop Dis 5, e1186, doi:10.1371/journal.pntd.0001186 (2011). 
60 
Gillespie, J. J. et al. A Rickettsia genome overrun by mobile genetic elements provides 
insight into the acquisition of genes characteristic of an obligate intracellular lifestyle. J 
Bacteriol 194, 376-394, doi:10.1128/JB.06244-11 (2012). 
61 
Rounds, M. A. et al. Identification of endosymbionts in ticks by broad-range polymerase 
chain reaction and electrospray ionization mass spectrometry. J Med Entomol 49, 843-
850 (2012). 
62 
Tokarz, R. et al. Virome analysis of Amblyomma americanum, Dermacentor variabilis, 
and Ixodes scapularis ticks reveals novel highly divergent vertebrate and invertebrate 
viruses. J Virol 88, 11480-11492, doi:10.1128/JVI.01858-14 (2014). 
63 
Kurtti, T. J. et al. Rickettsia buchneri sp. nov.: A rickettsial endosymbiont of the 
blacklegged tick Ixodes scapularis. Int J Syst Evol Microbiol, doi:10.1099/ijs.0.000047 
(2015). 
64 
Nakayama, Y. & Spielman, A. Ingestion of Lyme disease spirochetes by ticks feeding on 
infected hosts. J Infect Dis 160, 166-167 (1989). 
65 
Edlow, J. A. Tick paralysis. Curr Treat Options Neurol 12, 167-177, 
doi:10.1007/s11940-010-0068-7 (2010). 
66 
Gorman, R. J. & Snead, O. C. Tick paralysis in three children. The diversity of 
neurologic presentations. Clin Pediatr (Phila) 17, 249-251 (1978). 
67 
Ribeiro, J. M. et al. An annotated catalog of salivary gland transcripts from Ixodes 
scapularis ticks. Insect biochemistry and molecular biology 36, 111-129, 
doi:10.1016/j.ibmb.2005.11.005 (2006). 
68 
Van Nunen, S. A., O'Connor, K. S., Clarke, L. R., Boyle, R. X. & Fernando, S. L. An 
association between tick bite reactions and red meat allergy in humans. Med J Aust 190, 
510-511 (2009). 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
43
69 
Commins, S. P. et al. The relevance of tick bites to the production of IgE antibodies to 
the mammalian oligosaccharide galactose-alpha-1,3-galactose. J Allergy Clin Immunol 
127, 1286-1293 e1286, doi:10.1016/j.jaci.2011.02.019 (2011). 
70 
Commins, S. P. & Platts-Mills, T. A. Delayed anaphylaxis to red meat in patients with 
IgE specific for galactose alpha-1,3-galactose (alpha-gal). Curr Allergy Asthma Rep 13, 
72-77, doi:10.1007/s11882-012-0315-y (2013). 
71 
Brown, A. F. & Hamilton, D. L. Tick bite anaphylaxis in Australia. Journal of accident & 
emergency medicine 15, 111-113 (1998). 
72 
Valls, A., Pineda, F., Belver, M., Caballero, T. & Lopez Serrano, M. C. Anaphylactic 
shock caused by tick (Rhipicephalus sanguineous). J Investig Allergol Clin Immunol 17, 
279-280 (2007). 
73 
Acero, S., Blanco, R. & Bartolome, B. Anaphylaxis due to a tick bite. Allergy 58, 824-
825 (2003). 
74 
Kleine-Tebbe, J. et al. Bites of the European pigeon tick (Argas reflexus): Risk of IgE-
mediated sensitizations and anaphylactic reactions. J Allergy Clin Immunol 117, 190-195, 
doi:10.1016/j.jaci.2005.08.056 (2006). 
75 
Van Wye, J. E., Hsu, Y. P., Lane, R. S., Terr, A. I. & Moss, R. B. IgE antibodies in tick 
bite-induced anaphylaxis. J Allergy Clin Immunol 88, 968-970 (1991). 
76 
Burke, G. et al. Hypersensitivity to ticks and Lyme disease risk. Emerg Infect Dis 11, 36-
41, doi:10.3201/eid1101.040303 (2005). 
77 
Krause, P. J. et al. Dermatologic changes induced by repeated Ixodes scapularis bites and 
implications for prevention of tick-borne infection. Vector Borne Zoonotic Dis 9, 603-
610, doi:10.1089/vbz.2008.0091 (2009). 
78 
Alguire, P. C. & Mathes, B. M. Skin biopsy techniques for the internist. J Gen Intern 
Med 13, 46-54 (1998). 
79 
Aucott, J. N., Crowder, L. A. & Kortte, K. B. Development of a foundation for a case 
definition of post-treatment Lyme disease syndrome. Int J Infect Dis, 
doi:10.1016/j.ijid.2013.01.008 (2013). 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
44
APPENDIX 1: FOLLOWING PUNCH BIOPSY OF THE SKIN 
 
1. Keep the area dry and the dressing on for the first 24 hours. 
 
2. Thereafter, remove the dressing prior to showering. 
 
3. If adhesive steri-strips (which look like small pieces of tape) were used to close the 
incision, do not remove them. They will gradually fall off on their own. 
 
4. If sutures were placed, reapply Vaseline and a Band-Aid daily as needed. You will return 
4 to 7 days following the procedure for removal of the sutures.  
 
5. If you experience discomfort at the biopsy site, you can take acetaminophen (brand name: 
Tylenol), two 325 mg tablets every 4 to 6 hours as needed (do not take more than 12 
tablets in 24 hours) or ibuprofen (brand names: Advil, Motrin), one to two 200 mg tablets 
4 to 6 hours as needed (do not exceed 6 tablets in 24 hours). 
 
6. If the biopsy site begins to bleed, apply direct pressure for 10 minutes. If it continues to 
bleed, call us.  
 
7. Skin infection can follow any surgical procedure. If you develop increased pain, redness, 
pus or swelling at the biopsy site, call us. 
 
Contact numbers: 
Hospital  
 
 
 
Dr. 
 
 
 
________________ 
Page Operator  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
45
APPENDIX 2: CARE OF THE XENODIAGNOSIS SITE 
 
Instruction Sheet 
 
Tick placement and care of site: 
 
• Your dressing is to remain in place for 4 to 6 days. The dressing keeps the ticks in place. 
Please do not open or remove the dressing during the study period.  
• Do not scratch the dressing site if it itches. You may take oral diphenhydramine 
(Benadryl) for itching. 
• When showering, please cover the dressing site with AquaGuard. Remove the 
AquaGuard promptly after showering. Gently pat dry the dressing if necessary. Please 
check that the dressing is firmly in place. 
• Refrain from aerobic exercise or any activity that would induce heavy perspiration. 
• Please refrain from high-risk tick exposure activities (hiking, gardening, etc.) 
• Please do not take garlic supplements for the next 4 to 6 days. Garlic as seasoning in food 
is okay. 
• Check the dressing daily to make sure all edges are sealed. Call the study personnel if 
there is a problem with the dressing. You can contact _____________________ at phone 
number ____________________________. 
 
 
Diary Card: 
- Day 1 is the date ticks were placed. 
- Complete the diary card each day. 
- Please note any new medications taken during the study period. 
 
 
Scheduled visit date and time for tick 
removal:___________________________________________. 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
46
APPENDIX 3: TOXICITY GRADING SCALE 
 
(Adapted from Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent 
Volunteers Enrolled in Preventive Vaccine Clinical Trials (Food and Drug Administration, 
Center for Biologics Evaluation and Research, September 2007). 
 
Local Reaction 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Pain 
Does not interfere 
with activity 
Repeated use of 
non-narcotic pain 
reliever >24 hours 
or interferes with 
activity 
Any use of 
narcotic pain 
reliever or prevents 
daily activity 
Emergency room 
(ER) visit or 
hospitalization 
Tenderness 
Mild discomfort 
to touch 
Discomfort with 
movement 
Significant 
discomfort at rest 
ER visit or 
hospitalization 
Erythema/Redness 
2.5-5 cm 
5.1-10 cm 
>10 cm 
Necrosis or 
exfoliative 
dermatitis 
Induration/Swelling 
2.5-5 cm and 
does not interfere 
with activity 
5.1-10 cm or 
interferes with 
activity 
>10 cm or prevents 
daily activity 
Necrosis 
 
Vital Signs* 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life Threatening 
(Grade 4) 
Fever (°C) (°F) ** 
38.0-38.4 
100.4-101.1 
38.5-38.9 
101.2-102.0 
39.0-40 
102.1–104 
>40 
>104 
Tachycardia-beats 
per minute 
101-115 
116-130 
>130 
ER visit or hospitalization for 
arrhythmia 
Bradycardia-beats 
per minute*** 
50-54 
45-49 
<45 
ER visit or hospitalization for 
arrhythmia 
Hypertension 
(systolic)-mm Hg 
150-160 
161-170 
>170 
ER visit or hospitalization for 
malignant hypertension 
Hypertension 
(diastolic)-mm Hg 
95-100 
101-105 
>105 
ER visit or hospitalization for 
malignant hypertension 
Hypotension 
(systolic)-mm Hg 
85-89 
80-84 
<80 
ER visit or hospitalization for 
hypotensive shock 
* Subject should be at rest for all vital sign measurements. 
** If oral temperature; no recent hot or cold beverages or smoking. 
*** When resting heart rate is between 60-100 beats per minute. Use clinical judgment when 
characterizing bradycardia among some healthy subject populations, for example, conditioned 
athletes. 
 
 

 
 
Protocol Version 10 
22 Mar 2021 
 
 
47
 
Systemic 
(General) 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4) 
Nausea/vomiting 
No interference 
with activity or 1-
2 episodes/24 
hours 
Some interference 
with activity or >2 
episodes/24 hours 
Prevents daily 
activity, requires 
outpatient IV 
hydration 
ER visit or 
hospitalization for 
hypotensive shock 
Diarrhea 
2-3 loose stools 
or <400 gms/24 
hours 
4-5 stools or 400-
800 gms/24 hours 
6 or more watery 
stools or 
>800gms/24 
hours or requires 
outpatient IV 
hydration 
ER visit or 
hospitalization 
Headache 
Mild increase 
over baseline. 
Moderate increase 
over baseline, with 
repeated use of 
non-narcotic pain 
reliever >24 hours 
and some 
interference with 
activity 
Significant 
increase over 
baseline; any use 
of narcotic pain 
reliever or 
prevents ADL 
Disabling, ER visit 
or hospitalization 
Fatigue 
Mild fatigue over 
baseline 
Moderate fatigue 
over baseline or 
causing difficulty 
performing some 
activities of daily 
living (ADL) 
 
Severe fatigue 
over baseline, 
interfering with 
ADL 
 
Disabling, ER visit 
or hospitalization 
Myalgia 
Mild increase 
over baseline 
Moderate increase 
over baseline, or 
causing difficulty 
with ADL. 
Significant 
increase over 
baseline; 
interfering with 
ADL 
Disabling, ER visit 
or hospitalization