english: dr. liz zubek & dr. alison bested

Creation of standardized educational material for public health care

providers regarding the Prevention, Identification, Treatment and Management of Lyme disease

Educational Gaps

Alison C. Bested MD FRCPC Elizabeth Zubek BScMed MD CCFP FCFP

Federal Lyme Disease Conference Ottawa, Ontario

May 16, 2016

No disclosures

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1. Need - Clinical Case Definition to diagnose & treat patients with Lyme Disease/Tick Borne Diseases (LD/TBD) -Epidemiological Case Definition of Lyme Disease

2. Understand - Pathophysiology unclear: acute & chronic infection in LD

3. Need - Laboratory tests for acute LD - none 4. Need - Laboratory tests to monitor treatment,

relapse or re-infection LD 5. Need - enhanced Prevention/Education

Strategies

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4 Ogden, N. International Journal for Parasitology 2006

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Summer

Winter

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Scott, JD et al. Birds Disperse Ixodid (Acari: Ixodidae) and Borrelia Burgdorferi-Infected Ticks in Canada. J Med Entomology; 2001:38(4);493-500.

Rich Richter Dania, Spielman Andrew, Komar Nicholas, Matuschka Franz-Rainer,

Harvard School of Public Health. Competence of American robins as reservoir hosts for Lyme Disease spirochetes, Emerging Infectious Diseases, 03/01/2000

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Ticks Feeding on Dog Ear

• Time of attachment: unknown in humans • Animal models, Borrelia infection occur in <16 hrs • Spirochetes in tick salivary glands prior to feeding

may cause rapid Borrelia transmission from ticks to humans, case studies show rapid transmission

Cook MJ. Lyme borreliosis: a review of data on transmission time after tick attachment. Int J Gen Med. 2014 Dec. 19;8:1-8.

Hynote, ED et al. Clinical evidence for rapid transmission of Lyme disease following a tick bite. Diagnostic Microbiology and Infectious Disease. 2012:72:188-192.

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Bb - facultative anaerobe - micro-aerobic, low O2 tissues

e.g. connective tissue

Intracellular

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Immune-protected sites CNS, joints, eyes

Human tissue models Ma 1991 Endothelial cells Klempner 1993 Fibroblasts Girschick 1996 Synovial cells Dorward 1997 Lymphocytes Livengood 2006 Neurons and glial cells

Biopsy specimens MacDonald 1989 Neurons Aberer 1996 Dermatocyctes Miklossy 2008 Neurons Placenta 2006 Larrson

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Autoimmune Diseases and the Promise of Stem Cell-Based Therapies. In Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services, 2009. Figure 6.1. Immune Response to Self or Foreign Antigens. (© 2001 Terese Winslow)

Bb Immune Tactics that Avoid Our Defenses 1. Antigenic variation (VlsE locus in Bb) – change in protein coat

Antibody response a step behind 2. Immune Suppression 3. Physical Seclusion - Intracellular Sites - Extracellular Sites Secreted Factors 4. Inhibit complement binding

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VlsE types

Anti-VlsE antibodies

Norris SJ. Antigenic Variation Systems of Lyme Disease Borrelia: Eluding Host Immunity through both Random, Segmental Gene Conversion and Framework Heterogeneity. Microbiol Spectr. 2014 Dec;2(6). doi:10.1128/microbiolspec. MDNA3-0038-2

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2

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Serologic Laboratory Test (ELISA) - Supplemental to the clinical diagnosis of Lyme disease (history, tick exposure, physical findings) - Laboratory test is not the primary basis for making diagnostic or treatment decisions Rana Filfil, PhD, Health Canada, Canadian Adverse Reaction Newsletter. Volume 22:Number 4;October 2012

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• detect antibodies in blood

• Test for C6 peptide (VlsE gene) highly conserved 25 aa sequence)

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A: Patient T cells + Lyme antigens B: Cytokines secreted by activated T memory cells are captured C: Cytokine concentration measured with color reagent

Indirect Elispot

Western Blot: Bands specific for Lyme disease: Outer surface C 23 kDa, Outer surface A 31 kDa, Outer surface B 34 kDa, 39 and 83-93 • IgM: at least 2 of 3 bands positive

• IgG: at least 5 of 10 bands positive

• Better sensitivity than ELISA in early infection

• Canada: Doctors cannot order Western Blot - acute • The individual bands are not reported in Canadian

laboratory report • Canadian WB contains only 1/12 species of

Borrelia causing borreliosis in humans • How many patients have a false negative test

from 1/12 species tested?

IgG IgM

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Schutzer SE, Whole-genome sequences of thirteen isolates of Borrelia burgdorferi. J Bacteriol. 2011 Feb;193(4):1018-20. doi: 10.1128/JB.01158-10. Epub 2010 Oct 8.

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Magni R, Espina BH, Shah K, et al. Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis. Journal of Translational Medicine. 2015;13:346. doi:10.1186/s12967-015-0701-z.

OspA (Outer Surface Protein A) Common in all Borrelia species: Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia spielmanii, Borrelia bissettii

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Specificity %Healthy people identified as not being sick

99.5%

Sensitivity

%Sick people identified as sick

30%

True Positive Rate True Negative Rate

Testing for early Lyme Disease (patients with EM, unspecified no EM)

TESTS Sensitivity

Specificity

Two-Tier System (ELISA+WB)

35.0%

99.5%

C6 Peptide 66.5% 98.9%

ELISPOT European testing

84.0%

94.0%

NANOTRAP

100%

100%

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Clinical Diagnosis

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2016 PHAC Lyme Endemicity Map

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http://healthycanadians.gc.ca/diseases-conditions-maladies-affections/disease - maladie/lyme/risks- risques-eng.php#a3

Clusters of Symptoms that are not typical: • Fever • Rash • Flu-like symptoms • Eye pain, Bell’s palsy, burning sensations • Swollen joints/Arthritis • Mental confusion/Alzheimer’s • Mood changes: Anxiety or Depression • Heart rhythm changes

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24 Logigian, EL, Kaplan RF, Steere AC. Chronic neurological manifestations of Lyme disease. N Eng J Med 1990:323:1438-1444.

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***a high level of suspicion*** Persisting flu-like illness

Acute neurological disease Acute arthritis/arthralgias New carditis/arrhythmias

KB Liegner, MD Bernard Cohen; http://www.DermAtlas.org

M. Patmas

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All

E. Maloney

SmithKline Beecham Biologicals www.lyme.org accessed 4/11/07

SmithKline Beecham Biologicals www.lyme.org accessed 4/11/07

• Glaude study: tertiary rheumatology clinic • Only 37.5% of referring MDs suspected Lyme • Only 4/17 recalled tick bite • Only 3/17 had erythema migrans • 5/17 had neurological signs & symptoms • 2/17 had Abx - refractory Lyme arthritis

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Conclusion:

“Educational initiatives should be undertaken to increase local awareness of this treatable cause of arthritis in children.”

Glaude PD, Huber AM, Mailman T, Ramsey S, Lang B, Stringer E. Clinical characteristics, treatment and outcome of children with Lyme arthritis in Nova Scotia. Paediatr Child Health. 2015 Oct;20(7):377-80.

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West Coast Lyme Survey of MDs

1673 BC MDs surveyed: Knew erythema migrans is diagnostic for Lyme: 24.4% FPs, 28.2% specialists Would give antibiotics to a patient with an EM rash and no laboratory testing performed: 58.3% FPs, 54.8% specialists

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Knew patients with EM and negative test results do not need retesting (as EM is diagnostic): 6.8% FPs, 8.9% specialists Knew of possible co-infection with anaplasmosis (formerly human granulocytic ehrlichiosis) 10.1% FPs, 15.2% specialists

Henry B, Crabtree A, Roth D, Blackman D, Morshed M. Lyme disease: Knowledge, beliefs, and practices of physicians in a low-endemic area. Canadian Family Physician. 2012/05/01 00:00; 58(5): e289-e295 30

“The ER doctor told me Lyme was only East of the Rockies. They sent me home and told me to take aspirin for the flu. I knew there was something very different going on in my body.”

BC patient with Tick borne disease 31

Co-infections Tick-borne diseases are not confined to

Lyme Disease

237 bacterial genera commonly detected in unfed ticks, fed ticks, and rat blood samples after tick bites

Wormser GP 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. 2006 Nov 1;43(9):1089-134. Zhang XC, Yang ZN, Lu B, Ma XF, Zhang CX, Xu HJ. The composition and transmission of microbiome in hard tick, Ixodes persulcatus, during blood meal. Ticks Tick Borne Dis. 2014 Oct;5(6):864-70.

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Co-infections: clinical presentations suggest

etiologies “Gang warfare” analogy to treatment approach

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Anaplasma: the worst “flu” ever

• Acute onset fever, chills, headache

• Anorexia, cough, atypical pneumonitis

• Blood work +/- thrombocytopenia, leukopenia, increased Liver Function Tests

Dumler JS1, Madigan JE, Pusterla N, Bakken JS. Ehrlichiosis in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis. 2007 Jul 15;45 Suppl 1:S45-51.

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Bartonella: not an anxious somatizer Unusual neurological symptoms Anxiety, anger, irritability Eye pain or visual disturbances Striae (stretch marks) in odd places Deep bone pain, often the soles Lymphadenopathy Day sweats Bowel issues Costal margin pain Unusual rashes, vasoproliferative lesions Endocarditis/myocarditis

Striae

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Maggi RG, Mozayeni BR, Pultorak EL, Hegarty BC, Bradley JM, Correa M, Breitschwerdt EB. Bartonella spp. Bacteremia and Rheumatic Symptoms in Patients from Lyme Disease–endemic Region. Emerging Infectious Diseases. 2012/05/01 00:00; 18(5): 783-791 Harms A, Dehio C. Intruders below the Radar: Molecular Pathogenesis of Bartonella spp. Clinical Microbiology Reviews. 2012/01/01 00:00; 25(1): 42-78

Babesia: often overlooked and hard to prove

Drenching night sweats or fevers Chest wall pain Air hunger Headache at crown Vivid dreams

Mayne PJ. Clinical determinants of Lyme borreliosis, babesiosis, bartonellosis, anaplasmosis, and ehrlichiosis in an Australian cohort. International Journal of General Medicine. 1/01/01 00:00; 8: 15-26

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Chlamydia Pneumonia Reactive arthritis

Multiple-sclerosis-like Alzheimer's-like Chronic asthma Severe fatigue

Carter JD et al. Combination antibiotics as a treatment for chronic Chlamydia-induced reactive arthritis: a double-blind, placebo-controlled, prospective trial. Arthritis Rheum. 2010 May;62(5):1298-307. Miklossy J. Emerging roles of pathogens in Alzheimer disease. Expert Rev Mol Med. 2011 Sep 20;13:e30. Chia JK, Chia LY. Chronic Chlamydia pneumoniae infection: a treatable cause of chronic fatigue syndrome. Clin Infect Dis. 1999 Aug;29(2):452-3.

• Autoimmune illnesses • Chronic asthma • Arthralgia • Multiple neurological symptoms

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Waites KB, Talkington DF. Mycoplasma pneumoniae and Its Role as a Human Pathogen. Clin Microbiol Rev. 2004 Oct; 17(4): 697–728. Eskow E, Adelson ME, Rao RV, Mordechai E. Evidence for disseminated Mycoplasma fermentans in New Jersey residents with antecedent tick attachment and subsequent musculoskeletal symptoms. J Clin Rheumatol. 2003 Apr;9(2):77-87. Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev. 2008 Nov;32(6):956-73.

• One year history of escalation: waking from naps with pain in legs and feet, kicking and screaming “it’s broken!”. Pain 8/10

• Newly hyperactive, ++ volatile emotions • New behavior troubles 7/10 • Constant headache • one eyelid droops variably • No recollection of rashes or insect bites

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• CRP <0.3, RF <10 • ANA + speckled pattern, titre 160 • Minor elevation Alpha 2 globulin • Normal: CBC, liver, kidney, electrolytes • Mycoplasma IgM equivocal • Bartonella neg <1:64

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Borrelia: ELISA neg Western blot: neg C6 antibody: REACTIVE Immune blot for B garinii: neg Immune blot for B afzelii: neg

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• Maximal Ibuprofen & Tylenol not controlling his pain

• Pediatric neurology referral

• Elispot for Borrelia – Lyme disease

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• Borrelia burgdorferi fully antigen + 5

• Borrelia peptide mix + 5

• Borrelia LFA-1 < 2

Indicates immune cellular activity against Borrelia burgdorferi

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I found in his chart, while writing pediatric neurology consult: Previous Year: child walk-in visit for “insect bite … with large unusual local reaction”

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• Amoxil 50-100 mg/kg/day • Rifampin 10 mg/kg/day

Optometrist referral and then: • Plaquenil 100 mg BID • Probiotics 2 hours after Antibiotic

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• accidental double dosing:

Result: 2 days without pain and eye droop resolved! • Amoxil increased to 75 mg/kg/day

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• Decreased severity of pain • Some days “back to being Donny” • More good days than bad • Eye droop the telltale marker!

Risk/benefit analysis: informed decision to

continue antibiotics

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Happy, sparkling, interactive Extended family noted dramatic difference 11 days of lid droop gone Change in location of pain, from feet/toes to

knees Plan: try Azithromycin/Rifampin/Plaquenil

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Within 2.5 weeks eye droop, leg pain, crying/tantrums/anger & bedwetting returned

Response on restarting: partial

? Resistance or tolerance? Change to Septra/Amoxil/Azithromycin Naturopath for herbals: BLt tincture Within 4 weeks dramatic improvement

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Minor leg pain Rare day-end lid droop Scholastically excelling! Socially excelling!

Elispot 0 on all levels showing no T-immune cell response to Borrelia

Now: One year later improvement is sustained

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Educational Points from this Case:

• Initial MD missed EM (erythema migrans)

• Our tests “failed” without clinical aspect

• Treatment had to be individualized, flexible and “n of 1 trial”

• Life changing benefits from not giving up

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Why I refuse to call it Post Treatment Lyme

Educational Needs for

Tolerance and Persistence: The Importance of MDK

(Mean Duration for Killing)

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Brauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol. 2016 Apr 15;14(5):320-30.

• Able to switch between forms • Cell-wall deficient: Cyst or Round Body form

• Response to environmental changes • Potential significance

1) Enhanced survival - immune evasion, antibiotic tolerance 2) Serology criteria inapplicable

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Cyst or Round Body

Mobile Spirochete Intracellular

Spirochete /Cell Wall Intracellular

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Cyst /Round Body/Cell Wall

Deficient

Cell Wall Inhibitor Amoxicillin Cefuroxime

Cystic Drugs Metronidazole Plaquenil

Intracellular Zithromax Biaxin Doxycycline

Cameron DJ, Johnson LB, Maloney EL. Evidence assessments and guideline recommendations in Lyme disease: the clinical management of known tick bites, erythema migrans rashes and persistent disease. Expert Rev Anti Infect Ther. 2014 Sep; 12(9):1103-1135. doi:10.1586/14787210.2014.940900. Epub 2014 Jul 30.

• Stationary-phase in vitro Bb persisters that survived treatment with Doxycycline and amoxicillin

• Studied 1524 compounds • 165 had more activity than Doxycycline and

amoxicillin

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Feng J, Auwaerter PG, Zhang Y. Drug combinations against Borrelia burgdorferi persisters in vitro: eradication achieved by using Daptomycin, Defoperazone and Doxycycline. PLoS One. 2015 Mar 25;10(3):e0117207.

Daptomycin and Clofazimine had the highest activity on NON-GROWING persisters; POOR MIC (minimum inhibitory concentration) against GROWING Bb

“Daptomycin was the common element in the most active regimens” Daptomycin + Doxycycline + ß-lactams Daptomycin + Doxycycline + Clofazimine Daptomycin + Doxycycline + Cefoperazone

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• 90% had previous Antibiotics, median 2 courses and median > 1 month

• 2 weeks IV ceftriaxone gave a 5 point improvement on SF-36 physical scales

• Improvement continued for a year • Wings of study post-IV were single agent oral,

NOT combination therapy used by clinicians

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Berende A et al. Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. N Engl J Med. 2016 Mar 31;374(13):1209-20.

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• Diversity of presentation: Great Imitator • Variable morphologies: spirochete, round bodies, intracellular • Biofilms • Co-infections • Co-morbidities: impact on endocrine and immune systems

• Slow reproductive cycle, stationary phases • Other spirochete models (TB, syphilis) • Example: latent TB treated with 6-9 months INH or 3-4

months of combinations; ACTIVE TB treated with 4 to 7 months of combinations - PHAC

• Syphilis has 22 functioning genes, the Lyme spirochete has 132!

• “nearly all recommendations for the treatment of syphilis are based not only on clinical trials and observational studies, but many decades of clinical experience” - CDC.

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Awareness in Action

Education Needs:

1. Higher Level of Suspicion “Great Imitator”

2. Acute phase serology tests NOT helpful! Clinical diagnosis is key much more than “the target lesion”

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Lyme Treatment Essential Education

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Educate: Principles of Treatment LD

1. Individual Patient-centered

2. Challenging: must be an N-of-1 trial with informed consent of risks and benefits

3. Possibly complicated by co-infections

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• Wear hat, long sleeves and pants and light coloured clothing

• Tuck pant legs into socks • Use DEET insect repellant • Check for ticks on body after outside • Vaccinate pets and monitor for ticks • Safety Zone of no plants between

forest and property

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Treat with antibiotics: Doxycycline 100 -200 mg twice daily for 20 days to

prevent chronic disabling Lyme disease

Doctor will submit the tick to PHL for testing for Bb

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• Doctors nervous of “doing the right thing” because they fear reprisals

• Resulted in fewer doctors treating LD • Many patients must go to US for

treatment • Patients are driven underground to try

to find doctors who will treat them to help them get better

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Patients’ Perspective: Why are Canadian patients leaving the

country for treatment?

• US Doctors with Extensive Expertise

• They get better

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Diagnosis/management of Lyme hampered by: • Incomplete understanding of Borrelia’s

pathophysiology • Lack of reliable biomarkers • Insufficient clinical trial evidence

In the future, successful management will be based • Identification of actual pathophysiologic

mechanisms • Methodology for determining which bacteria are

operating in a specific patient • Targeted therapies, including combinations of

therapeutic modalities

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• Create LD medical undergraduate curricula

• Accredited online courses from CFPC, RCPS, CMA and Provincial Colleges

• Create Subspecialty post-graduate training in tick-borne illness available for physicians in any field (family practice, rheumatology, psychiatry, neurology, cardiology, general internal medicine etc.)

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Research – better clinical tests based on Borrelia species (burgdorferi, miyamotoi etc.) pathophysiology Educate - Doctors about clinical guidelines - Public about prevention Support - chronically ill patients Support - doctors treating LD in Canada Creation of medical experts in emerging medical field of tick borne illnesses Creation of funded National Lyme Network including:

researchers, clinicians and patients to formulate next steps quickly

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Cost Effectiveness

Weeks vs. Years Treatment

Ability vs. Chronicity

Australia’s skin cancer prevention slogan: Sid the Seagull Slip on a shirt, slop on sunscreen, slap on a hat & Stop skin cancer.

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Cover up from your head to your feet

Spray with DEET When ticks eat your meat Treat Prevent Lyme Disease!

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Adelson ME, Rao RVS, Tilton RC, et al. Prevalence of Borrelia burgdorferi , Bartonella spp., Babesia microti, and Anaplasma phagocytophilum in Ixodes scapularis ticks collected in northern New Jersey . J Clin Microbio 2004; 42: 2799-2801.

Aguero-Rosenfeld ME. Laboratory aspects of tick-borne diseases: Lyme, human granulocytic ehrlichiosis and babesiosis. Mt Sinai J Med. 2003; 70(3):197-206.

Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev. 2008 Nov;32(6):956-973.

Autoimmune Diseases and the Promise of Stem Cell-Based Therapies . In Stem Cell Information. National Institutes of Health, U.S. Department of Health and Human Services, 2009. Figure 6.1. Immune Response to Self or Foreign Antigens. Schematic Terese Winslow

Berende A et al. Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. N Eng J Med. 2016 Mar 31;374(13):1209-1220.

Breitschwerdt EB, Maggi RG, Nicholson WL, Cherry NA, Woods CW. Bartonella sp. Bacteremia in Patients with Neurological and Neurocognitive Dysfunction. J Clin Microbio 2008; :2856-2861.

Brauner A, Fridman O, Gefen O, Balaban NQ. Distinguishing between resistance, tolerance and persistence to antibiotic treatment. Nat Rev Microbiol. 2016 Apr 15;14(5):320-330.

Brorson O; Brorson S. A Rapid Method for Generating Cystic Forms of Borrelia burgdorferi, and Their Reversal to Mobile Spirochetes. APMIS 1998; 106(12):1131-1141.

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Carter JD et al. Combination antibiotics as a treatment for chronic Chlamydia-induced reactive arthritis: a double-blind, placebo-controlled, prospective trial. Arthritis Rheum. 2010 May;62(5):1298-1307.

Chia JK, Chia LY. Chronic Chlamydia pneumoniae infection: a treatable cause of chronic fatigue syndrome. Clin Infect Dis. 1999 Aug;29(2):452-453.

Chandra A, Wormser GP, Klempner MS, Trevino RP, Crow MK, Latov N, Alaedini A. Anti-neural antibody reactivity in patients with a history of Lyme borreliosis and persistent symptoms. Brain Behav Immun. 2010 Aug;24(6):1018-24. Clark K. Borrelia species in host-seeking ticks and small mammals in northern Florida. J Clin Microbiol. 2004 Nov;42(11):5076-5086.

Clark KL, Leydet B, Hartman S. Lyme Borreliosis in Human Patients in Florida and Georgia, USA. Int J Med Sci 2013;10(7):915-931.

Cook MJ. Lyme borreliosis: a review of data on transmission time after tick attachment. International Journal of General Medicine. 2015;8:1-8. doi:10.2147/IJGM.S73791.

DeMartino SJ, Carlyon JA, Fikrig E. Coinfections with Borrelia burgdorferi and the agent of human granulocytic ehrlichiosis. N Engl J Med 2001; 345:150-151.

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Des Vignes F, Piesman J, Heffernan R, Schulze T, Stafford K, Fish D. Effect of tick removal on transmission of Borrelia burgdorferi and Ehrlichia phagocytophila by Ixodes scapularis nymphs. J Infect Dis. 2001;183:773-778.

Dumler JS, Bakken JS: Human granulocytic ehrlichiosis in Wisconsin and Minnesota: A frequent infection with the potential for persistence. J Infect Dis 1996;1 73:1027-1030.

Dumler JS1, Madigan JE, Pusterla N, Bakken JS. Ehrlichiosis in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis. 2007 Jul 15;45 Suppl 1:S45-51.

Embers ME, Barthold SW, Borda JT, et al. Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.PLoS One. 2012;7(1):e29914. Epub 2012 Jan 11. Erratum in: PLoS One. 2012;7

Embers ME, Ramamoorthy R, Philipp MT. Survival strategies of Borrelia burgdorferi, the etiologic agent of Lyme disease Microbes and Infection 2004; 6:312-318.

Eskow E, Rao RV, Mordechai E. Concurrent infection of the central nervous system by Borrelia burgdorferi and Bartonella henselae: evidence for a novel tick-borne disease complex. Arch Neurol 2001; 58:1357-1363.

Eskow E, Adelson ME, Rao RV, Mordechai E. Evidence for disseminated Mycoplasma fermentans in New Jersey residents with antecedent tick attachment and subsequent musculoskeletal symptoms. J Clin Rheumatol. 2003 Apr;9(2):77-87.

Feng J, Auwaerter PG, Zhang Y. Drug combinations against Borrelia burgdorferi persisters in vitro: eradication achieved by using Daptomycin, Defoperazone and Doxycycline. PLoS One. 2015 Mar 25;10(3):e0117207.

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Falco RC, Fish D, Piesman J. Duration of Tick Bites in a Lyme Disease-endemic Area. Am J Epidemiol 1996;143(2):187 -19Frank C, Fix AD, Peña CA, Strickland GT. Mapping Lyme Disease incidence for diagnostic and preventive decisions. Emerg Infect Dis. 2002;8(4):427-429.

Girschick HJ, Huppertz HI, Russmann H, Krenn V, Karch H. Intracellular persistence of Borrelia burgdorferi in human synovial cells. Rheumatol Int 1996; 16:125-132.

Glaude PD, Huber AM, Mailman T, Ramsey S, Lang B, Stringer E. Clinical characteristics, treatment and outcome of children with Lyme arthritis in Nova Scotia. Paediatr Child Health. 2015 Oct;20(7):377-380.

Harms A, Dehio C. Intruders below the Radar: Molecular Pathogenesis of Bartonella spp. Clinical Microbiology Reviews. 2012/01/01 00:00; 25(1): 42-78

Henry B, Crabtree A, Roth D, Blackman D, Morshed M. Lyme disease: Knowledge, beliefs, and practices of physicians in a low-endemic area. Canadian Family Physician. 2012/05/01 00:00; 58(5): e289-e295

Hojgaard A, Eisen RJ, Piesman J. Transmission dynamics of Borrelia burgdorferi s.s. during the key third day of feeding by nymphal Ixodes scapularis (Acari: Ixodidae). J Med Entomol. 2008; 45(4):732-736.

Holden K, Hodzic E, Feng S, Freet KJ, Lefebvre RB, Barthold SW. Coinfection with Anaplasma phagocytophilum alters Borrelia burgdorferi population distribution in C3H/HeN mice. Infect Immun. 2005 Jun;73(6):3440-3444.

http://healthycanadians.gc.ca/diseases-conditions-maladies-affections/disease - maladie/lyme/risks- risques-eng.php#a3

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Hovius JWR, van Dam AP, Fikrig E. Tick–host–pathogen interactions in Lyme borreliosis. Trends Parasitol 2007; 23(9):434-438.

Hynote, ED et al. Clinical evidence for rapid transmission of Lyme disease following a tick bite. Diagnostic Microbiology and Infectious Disease. 2012:72:188-192.

Jin C. Et al. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi. Cells 2013, 2, 607-620; doi:10.3390/cells2030607

Kersten A, Poitschek C, Rauch S, Aberer E. Effects of Penicillin, Ceftriaxone, and Doxycycline on Morphology of Borrelia burgdorferi. Antimicrob Agents Chemother 1995; 39(5):1127-1133.

Knauer J, Krupka I, Fueldner C, Lehmann J, Straubinger RK. Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model. J Antimicrob Chemother 2011;66(12):2814-1122.

Krause PJ, McKay K, Thompson CA et al. Disease-specific diagnosis of coinfecting tickborne zoonoses: babesiosis, human granulocytic ehrlichiosis, and Lyme disease. Clin Infect Dis 2002; 34:1184-1191.

Krause PJ, Spielman A, Telford SR 3rd, Sikand VK, McKay K, Christianson D, Pollack RJ, Brassard P, Magera J, Ryan R, Persing DH. Persistent parasitemia after acute babesiosis. N Engl J Med 1998; 339(3):160-165.

Krause PJ, Telford SR, Spielman A, et al: Concurrent Lyme disease and babesiosis: Evidence for increased severity and duration of illness. JAMA 1996; 275:

1657-1660.

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Larsson C. et al. Complications of Pregnancy and Transplacental Transmission of Relapsing-Fever Borreliosis. J Inf Dis 2006; 94(10):1367-1374.

Liang FT, Brown EL, Wang T, Iozzo RV, Fikrig E. Protective niche for Borrelia burgdorferi to evade humoral immunity. Am J Pathol 2004;165:977-985.

Liang FT, Jacobs MB, Bowers LC, Philipp MT. An immune evasion mechanism for spirochetal persistence in Lyme borreliosis. J Exp Med 2002;195:415-422.

Lucey D, Dolan MJ, Moss CW, et al. Relapsing illness due to Rochalimaea henselae in immunocompetent hosts: implication for therapy and new epidemiological associations . Clin Infect Dis 1992; 14:683-688.

Logigian, EL, Kaplan RF, Steere AC. Chronic neurological manifestations of Lyme disease. N Eng J Med 1990:323:1438-1444.

Magni R, Espina BH, Shah K, et al. Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis. Journal of Translational Medicine. 2015;13:346. doi:10.1186/s12967-015-0701-z.

MacDonald AB, Miranda JM. Concurrent neocortical borreliosis and Alzheimer’s disease. Hum Pathol. 1987; 18(7):759-761.

Maloney EL. The management of Ixodes scapularis bites in the upper Midwest. WMJ. 2011 Apr;110(2):78-81; quiz 85.

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