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Current State of Lyme diagnostics in Canada
Todd F. Hatchette MD FRCPC
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
Mitigating Potential Bias
• Funding for research program / honorarium has nothing to do with Lyme disease
Definitions
• Early Localized Lyme disease • Early Disseminated Lyme disease • Late Lyme disease • Post Lyme Disease Syndrome • Chronic Lyme disease
Further discussed by Bill Bowie
Early and Late Symptoms of Lyme Disease
Days to weeks
Weeks to months
Months to years
Lyme Disease Clinical Course Acute cutaneous
disease
Early disseminated disease
Late disseminated disease
http://ocw.jhsph.edu/courses/EpiInfectiousDisease/PDFs/EID_lec15_Griffin.pdf
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
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
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
• 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)
Performance of Serology Depends On The Stage Of The Disease
• 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
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
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
Performance of Serology Depends On The Stage Of The Disease
* Sera obtained after abx treatment
Molins et al 2014 JCM 52:10
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.
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
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
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
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
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.
• 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
Test performance as reported by Fallon in Clinical Infectious Diseases 2014
100 Alternative Lyme Tests
Why Specificity is as Important as Sensitivity
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).
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
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
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.
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
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
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
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