The document discusses the current state of Lyme disease diagnostics in Canada, detailing the various stages of the disease and how diagnostic accuracy is influenced by factors such as specimen type and methodology. It highlights the performance of serological tests, the challenges in diagnosing early infections and reinfections, and the variability in test results due to geographical diversity of the Lyme bacteria. Additionally, it emphasizes the need for improved testing approaches and better-coordinated research efforts to enhance diagnostic capabilities.

1. Current State of Lyme diagnostics
in Canada
Todd F. Hatchette
MD FRCPC

2. Faculty/Presenter Disclosure
• Faculty: Todd F. Hatchette
• Relationships with commercial interests:
– Grants/Research Support: Investigator collaborative research
agreements – anchor laboratory for the Severe Outcomes Surveillance
Network looking at influenza vaccine effectiveness and
pneumocococus in community acquired pneumonia (GSK/Pfizer)
– Speakers Bureau/Honoraria: Honorarium for a talk on latent TB
infection and reactivation with patients on biological therapies
(Abbvie)
– Consulting Fees: None
– Other: None

3. Mitigating Potential Bias
• Funding for research program / honorarium has nothing to do
with Lyme disease

4. Definitions
• Early Localized Lyme disease
• Early Disseminated Lyme disease
• Late Lyme disease
• Post Lyme Disease Syndrome
• Chronic Lyme disease
Further discussed by Bill Bowie

5. Early and Late Symptoms of Lyme
Disease
Days to weeks
Weeks to months
Months to years

6. Lyme Disease Clinical Course
Acute cutaneous
disease
Early disseminated
disease
Late
disseminated
disease
http://ocw.jhsph.edu/courses/EpiInfectiousDisease/PDFs/EID_lec15_Griffin.pdf

7. Factors that affect Diagnostic
accuracy
• Type of specimens
• Collection time with respect to disease state
• Kinetics of antibody expansion
• Methodology (i.e. molecular, cultivation,
serology, etc)
• Bacterial strains
– Travel History
• Prevalence

8. Laboratory Diagnosis of Lyme
disease
• Serology (two tier algorithm)
– EIA
• Whole cell EIA
• VlsE C6 peptide
• VIsE1 / C10 peptide
– Western Blot
• PCR
– For synovial fluid or CSF
• Culture
– Up to 50% in EM
• Tests with uncertain
performance
– Antigen detection
– Immunologic markers
• CD57 – NK cells
• Elispot

9. Two Tier Tesing
• Western blots can not be used independent of
EIAs
– EIAs are quantitative, blots are subjective
– IgM western blots have poor specificity. Only
diagnostic if used in first 6 weeks of infection
– European species can be falsely negative on NA
WB
Aguero-Rosenfeld and Wormser, 2015. Exprt Rev Mol Diagn 15:1-4
DeBiasi. 2014. Curr Infect Dis Rep 16:450-455

10. • Individual proteins differ widely in their sensitivity and specificity in
predicting final IgG WB result
• P39 and VlsE most consistent but ROC of these is < 90%
• No one protein is >90% predictive (ROC >90%) of IgG WB result
• Reducing level of response needed for a band to be considered
positive slightly increases sensitivity but decreases specificity
Individual proteins differ widely in their sensitivity
and specificity in predicting final IgG WB result
(Source R. Lindsay NML)

11. Performance of Serology Depends On The
Stage Of The Disease

12. • 1999-2001 East Lyme Connecticut
• 76 patients who had culture confirmed EM
• 147 with late Lyme or other illnesses
– objective clinical findings and positive serology
• PLD (Post LD or Chronic LD)
– pain or neurocognifive or fatigue < 6 months after
appropriate ABX Rx
• Controls
CID 2008 47:188-195

13. Performance of Serology Depends On
The Stage Of The Disease
Disease State C6 EIA EIA +
IgM WB
EIA +
IgG WB
EIA and
IgM or IgG
WB
Acute (stage 1) 19-38% 11-38% 6-15% 17-43%
Convalescent
after antibiotics
47-63% 39-70% 17-20% 53-75%
Disseminated
infection (acute
neurologic or
arthritis) (stage 2)
100% 85% 85% 100%
Persistent
infection (stage 3)
100% 23% 100% 100%
CID 2008 47:188-195

14. Disease State C6 EIA EIA +
IgM WB
EIA +
IgG WB
EIA and IgM
or IgG WB
PLDS (n=14) 43% 50% 36% 71%
Not Lyme (n=75)
(CFS, FM,MS,RA)
1% 0% 0% 0%
Healthy (endemic
area) (n=86)
5% 1% 1% 2%
Healthy (non-
endemic area)
(n=50)
2% 0% 0% 0%
Specificity of Two Tier Algorithm is High
CID 2008 47:188-195

15. Performance of Serology Depends On The
Stage Of The Disease
* Sera obtained after abx treatment
Molins et al 2014 JCM 52:10

16. New antigenic Targets
• Antigens expressed early in mammalian infection
– C6, a conserved 26 amino acid peptide within the Variable
major protein-like sequence (VlsE1)
– recombinant VlsE1 itself
– pepC10, a conserved 10 amino acid peptide portion of
OspC
• Immune response to VlsE1 are IgG mediated, even in
early disease, while pepC10 generates an early and
sometimes lasting IgM response (2, 5, 28).
• B. burgdorferi lipoprotein BBK07 – an
immunodominant antigen, is an in vivo-induced
surface antigen which is selectively expressed during
mammalian infection
Porwancher et al., Clin. Vaccine Immunol. 2011 18:851-859
Coleman et al., Clin Vaccine Immunol. 2011 18:406–413.

17. Assays Sensitivity (%) specificity
Early Early
disseminated
Late Healthy Patients with
non-LD
infection
WCS ELISA 74 97.7 98.4 96.4 89.3
WCS ELISA +
WB
35.2 77.3 95.9 99.5 99.2
C6 ELISA 66.5 88.6 98.4 98.8 99.5
C6 ELISA + WB 34.5 75 95.1 99.5 99.5
VlsE CIA 69.8 100 100 99.5 93.7
PepC10
kELISA (IgM)
47.3 46.1 10.3 100 98.0
VlsE/PepC10
kELISA
67.2 88.5 94.1 99.2 96.7
Evolution of EIAs: improved Sensitivity
Adapted from Theel S JCM 2016

18. Alternative algorithms
Whole cell EIA + C6 vs Current two tier
• Better sensitivity in early infection
Branda et al. Clinical Infectious Diseases 2011;53(6):541–547
• Specificity using healthy controls were equal

19. Alternative algorithms
VlsE1-IgG and pepC10-IgM
• second-tier: immunoassay using a multiplex microsphere system
that measures VlsE1-IgG and pepC10-IgM
• Better sensitivity in early infection
Porwancher et al., Clin. Vaccine Immunol. 2011 18:851-859

20. Summary of Lyme Disease Testing in Provinces
Prov. Tier 1 Tier 2
BC Zeus VlsE + pepC10 EIA
MarDx Western Blots (IgM & IgG), refer
European WB testing to NML
AB Immunetics C6 IgG/IgM ELISA refer all WB testing to NML
SK Diasorin Liason CLIA (IgG/IgM + VlsE), refer all WB testing to NML
MB Immunetics C6 IgG/IgM ELISA refer all WB testing to NML
ON Immunetics C6 IgG/IgM ELISA
MarDx Western Blots (IgM & IgG), refer
European WB testing to NML
QC Various* ELISA's to screen refer all WB testing to NML
NB Immunetics C6 IgG/IgM ELISA refer all WB testing to NML
NS
ZEUS WC IgG/IgM ELISA then
Immunetics C6 IgG/IgM ELISA,
refer all WB testing to NML
PE Referred to NS for testing
NL Referred to NML for all testing
NML Immunetics C6 IgG/IgM ELISA,
Euroimmun US (IgM & IgG)
Euroimmun European (IgG) Western Blots
* Immunetics C6 IgG/IgM ELISA, VIDAS Lyme IgG II EIA, Euroimmun Anti- Borrelia IgM and Anti-
Borrelia VlsE IgG ELISA

21. Alternative Methods can Lead to Spurious Results
• Fallon et al., 2014. Clin Infect Dis 59(12):1705–10
• In-house laboratory criteria for a positive IgM WB at Specialty Laboratory B were ≥2 of the following bands:
23–25, 31, 34, 39, 41, 83/93. Criteria for a positive IgG WB were ≥2 of the following bands: 23–25, 31, 34, 39,
41, 83/93.

22. • Compared the ability of Light Microscopy to
diagnose Borrelia infection in blood
– 25/41 healthy controls had positive microscopy
• PCR was not consistent between 4
laboratories and some concern about
contamination between positive controls
Infectious Disease 2016 48:411-419

23. Test performance as reported by Fallon in Clinical Infectious Diseases 2014
100
Alternative
Lyme Tests
Why Specificity is as Important as Sensitivity

24. Alternative Approach – Metabolomics
Molins et al., Clin Infect Dis 2015;60(12):1767–75
• Small molecule metabolites extracted from of
sera with methanol and analyzed by Liquid
Chromatography-Mass Spectrometry (LC-MS).

25. Diagnosis of Re-infection Is A Challenge
• There is no test of cure
• Re-infection identified in 5 prospective studies of
Lyme disease in the US
– Rate of re-infection/year - 1.2 – 3.1%
– Usually EM at a different site
• No pattern has been identified to differentiate re-
infection from previous infections
• Seroconversion or a 4x rise in titre could indicate
re-infection – difficult to do
Nadelman and Wormser, 2007 CID 45:1032-1038
Krause et al., 2006 AJTMH 75:1090-1094

26. Biodiversity
Cerar T 2016 EID 2016
• B. burgdoferi between Europe and
USA are different
• Clinical presentation between Europe
and USA are different
• More diversity in USA based on ospC

27. Strain Diversity Exists
Haninkova et al. Plos one 2013. 8(9)
Unrooted ML tree of B. burgdorferi based
on concatenated sequences of eight
MLST housekeeping genes
Venn diagrams depicting the geographical
distribution of B. burgdorferi in Lyme
disease patients.

28. Biodiversity affects the WB results
but less so for C6 or whole cell EIA
Wormser et al., Clinical Infectious
Diseases 2008; 47:910–4
RST OMP C

29. Diagnostic Challenges
Summary
• Poor performance of serology in early infection
• Seroconversion may not occur with early treatment
• No test of cure –
• serology can persist for a decade
• Diagnosis of re-infection is a challenge
• Influence of biodiversity needs to be explored further
• No current diagnostic testing for PLDS

30. Urgent Need for Well
Characterized Samples
• Convenient samples are used for determining
sensitivity and specificity
• Difficult to compare new tests if reference is based
on 2-tier serological results
• Unable to easily study bacteria by molecular
methods
• CDC does have a serum repository and some access
to that panel is offered to Canada
– Limited resource
– Better to have Canadian context

31. Diagnostic Challenges
Summary
• We need better defined cohorts to have
specimens to validate new methods as they
get developed
– Early Localized Lyme disease (30 days)
– Early Disseminated Lyme disease ( < 3 months)
– Late Lyme disease (> 3 months)
– Post Lyme Disease Syndrome
– Chronic Lyme disease
• Diagnosis based solely on clinical features
• Diagnosed with alternative testing methods