Xenodiagnosis After Antibiotic Treatment for Lyme Disease

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 posttreatment 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.

- 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 .

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 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

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 arthritis 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 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 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. 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.

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.

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).

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.

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.