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Understanding Laboratory Testing
for Lyme Disease
Photo credit: CDC PHIL
Christina Nelson, MD, MPH, FAAP
Medical Epidemiologist
Lyme Disease Clinician Forum
June 6th, 2013
National Center for Emerging and Zoonotic Infectious Diseases
Division of Vector-Borne Diseases
Disclosures
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No financial interests or relationships to disclose
Objectives
Discuss:
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Background on Lyme disease testing
Details of two-tier serologic testing
Utility of additional diagnostic tests (PCR, etc.)
“Alternative” laboratory tests
FAQs related to testing
Resources for clinicians
NOTE: Cases are reported based on patient's county of residence,
which may be different from where they were infected.
Background on Lyme Disease Testing
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The challenge: B. burgdorferi cannot be easily detected
from infected patients
o
Low # of spirochetes in blood, transient
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PCR detects Bb in blood of < ½ of patients during acute
dissemination (spirochetemic) phase of infection
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Spirochetes can be recovered from skin biopsy
specimens, but this is invasive
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Therefore, indirect methods must be employed to
detect infection  enter serology
Serologic Tests
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Detect the body’s immune response to an infection
Used for HIV, syphilis, viral hepatitis, etc.
“Window period” shortly after infection
Research has identified antibodies with the highest
sensitivity & specificity for evidence of Lyme disease
Dearborn meetings  consensus on approach to testing
and which antibodies (bands) to include
CDC. Recommendations for test performance and interpretation from the Second National Conference on
Serologic Diagnosis of Lyme Disease. MMWR 1995;44(31):590-1.
Engstrom et al. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol.
1995 ;33(2):419-27.
1995; 44(31): 590-1
When to Test?
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Testing is not required for patients with EM in
high incidence areas
o
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In such cases of early Lyme disease, clinicians can diagnose
clinically and begin treatment without confirmatory testing
In all other cases, laboratory testing is necessary
for diagnosis of Lyme disease
o
o
Atypical early disease
Bell’s palsy, arthritis, meningitis, etc.
Approach to Patient with Possible Early LD
Suspect Case
Consistent with EM + endemic region
Treat
Possible EM, Bell’s palsy, arthralgia, HA, or
other sign/symptom + endemic region
Other s/sx or history
suspicious for LD
No other s/sx or
nonspecific s/sx
Treat
+ test acute &
convalescent
Test acute &
convalescent
+/- treat
Adapted from: Goldsmith et al. Fitzpatrick’s Dermatology in General Medicine, 8th edition.
Nuts & Bolts of
Serologic Testing for Lyme Disease
Sensitivity of Two-Tier Serologic Testing
Lyme Disease Stage
EM rash (acute)
EM rash (convalescent)
Early neurologic
Late neurologic
Arthritis
Sensitivity (%)*
38
67
87
100
97
*Specificity of two-tier testing is generally > 95%
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Testing of patients with EM not generally necessary
Good sensitivity in later stages of disease
Bacon et al. JID 2003; 187:1187–99
Interpreting the Western Blot
IgM
Positive test
requires at least 2
out of 3 bands!
IgG
Positive test
requires at least 5
out of 10 bands!
Note:
p41 = flagellin.
Present in 1/3-1/2
of all healthy
people.
Aguero-Rosenfeld, et al. Diagnosis of Lyme Borreliosis. Clin Micro Rev 2005; 18: 484-509.
False Positive IgM Western Blots
kD
1 2 3 4
Columns:
1 = band locator
2 = intensity cutoff
3 = positive control
4 = patient sample. Band intensities below
cutoff or wrong position. NEGATIVE.
Fla 41
BmpA 39
OspC 23
50/182 (27%) IgM WB found to be false positive
for the following reasons:
• 45 / 50 had symptoms > 4 weeks
• 20 / 50 did not have first tier test +
• 6 / 50 scored incorrect bands
Seriburi et al. High frequency of false positive IgM immunoblots for
Borrelia burgdorferi in clinical practice. Clin Microbiol Infect 2011.
Expanding Antibody Profiles
IgG
IgM
1. Band locator
1. Band locator
2. Early localized
with EM
3. Early dissemin.
with multiple
EMs
2. Early dissem.
with neuro
involvement
3. Late dissem.
with arthritis
4. OspA vaccinee
(31 kDa)
Aguero-Rosenfeld et al., 2005. Diagnosis of Lyme Borreliosis. Clin Microbiol. Rev. 18:484-509
Lyme Serology Cross-Reactions
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Other spirochetes
• Tick-borne relapsing fever (Borrelia hermsii), syphilis
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Anaplasmosis
Leptospirosis
Autoimmune disorders – lupus, rheumatoid arthritis, etc.
Bacterial endocarditis
Epstein-Barr virus
Cross-reactions occur most often with EIA and IgM
o
IgG is more specific
Take-Home Points:
Appropriate Use of Testing
Haiku to Lyme Disease Testing
Where disease is rare
Positives mostly deceive
Even with good tests
IgM: Only for Early Lyme Disease
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If signs or symptoms > 30 days, do not order IgM
Western blot (second tier) test  only IgG
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Caution: some labs perform both IgM and IgG Western
blot as default
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May need to specifically request that IgM not be done
o
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Call lab or write instructions on order form
If not possible, at a minimum discount IgM results
Bottom Line on Serology
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For patients with classic EM who live in or traveled to high
incidence areas, laboratory testing is not necessary
o
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Two-tier serology is accurate when used appropriately
o
o
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Clinicians may diagnose clinically and begin treatment
Sensitivity and specificity are very good in disseminated disease
Avoid “shotgun” testing (low pre-test probability)
Limitations: low sensitivity in early disease, cross-reaction,
antibody persistence
Additional Diagnostic Tests
B. burgdorferi Culture
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Slow growth (days to weeks)
Cultivable human samples:
EM biopsy > blood > synovial tissue > CSF
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Growth is detected by direct visualization
o
o
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Dark-field microscopy or fluorescent staining  false-positive
reads possible from thread-like cellular debris, etc.
If visualized, should confirm organism with PCR
Bottom line: Valuable clinical and biological research
foundation but limited diagnostic utility
Aguero-Rosenfeld ME, Wang G, Schwartz I, Wormser GP. Diagnosis of Lyme borreliosis. Clin Microbiol Rev. 2005;18:484-509.
PCR
Synovial fluid may be positive in pts with Lyme arthritis
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o
CSF positive in ~30% of pts with early neuroborreliosis
Non-standardized, variety of approaches & targets
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However, mRNA (marker of spirochete viability) not detectable
No FDA cleared PCR assay for Lyme disease
Caveats: Highly sensitive so prone to contamination, does
not distinguish between living and dead organisms
Bottom line: Valuable research tool, may be useful in
unique clinical situations
Aguero-Rosenfeld et al. Diagnosis of Lyme borreliosis. Clin Microbiol Rev. 2005;18:484-509.
Li et al. Burden and viability of Borrelia burgdorferi in skin and joints of patients with erythema migrans or lyme arthritis..
Arthritis Rheum. 2011;63(8):2238-47.
CSF Testing for Neuroborreliosis
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Measure CSF/serum index of antibodies to Bb
o
o
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Differential antibody expression in CSF vs. serum may occur
o
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ELISA and/or Western blot
CSF and serum should be drawn simultaneously if possible
e.g. OspA & OspB antibodies are expressed more in CNS
PCR usually low-yield except in early neuroborreliosis
C6
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Vmp-like sequence expressed (VlsE) is an outer surface
lipoprotein of Bb
o
o
C6 is a highly conserved portion of VlsE’s region 6
Highly immunogenic
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C6 EIA is already FDA approved as a first-tier test
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Advantages over whole-cell sonicate EIA:
o
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Better marker of early disease
Improved sensitivity for European Lyme dz
C6 antibodies fade faster
Can parts of the two-tier approach be modified to simplify
and improve accuracy in early disease?
C6 As Second Tier?
Standard 2-tier
2-EIA
C6 EIA alone
2-EIA strategy realizes sensitivity benefits of C6 in early disease while
minimizing subjectivity and maintaining specificity of standardized 2-tier
Branda JA et al. 2011 Clin Inf Dis. 53:541-547
Alternative Tests for Lyme Disease
“In-house” Assays
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Developed by private laboratories
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Not sold or distributed across state lines
o
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Therefore FDA clearance not required
Clinical Laboratory Improvement Amendments
(CLIA) require specific record-keeping & tests for
analytic accuracy
o
Evaluation of clinical sensitivity and specificity not required
Alternative Tests for Lyme Disease
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1.
2.
Several found to be unreliable
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IGenex LUAT urine antigen test 1
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Phillips culture media 2
Klempner, MS et al. 2001 “Intralaboratory reliability of serologic and urine testing for Lyme disease.”
American Journal of Medicine 110:217-19).
Marques, AR et al. 2000 “Evaluation of a new culture medium for Borrelia burgdorferi.” Journal of Clinical
Microbiology 38:4239-41, enclosure 58; Tilton, RC et al. 2001 “Culture of Borrelia burgdorferi.” Journal of
Clinical Microbiology 39:2747).
Alternative “Culture” Test
Limitations and Incongruities of
New Culture Report
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Patients and illness poorly described
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Nested PCR controls: B. burgdorferi, B. garinii, or B. afzelii
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Among the 51 samples positive by PCR:
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40% had sequences identical to laboratory strain of B. burgdorferi
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> 41% identified as B. garinii or B. afzelii, strains not linked to
human illness acquired in the US
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All three genospecies were used as controls in the nested PCR
reactions
New Culture Technique for Lyme Disease
Pending further validation, health departments
should not consider results generated with this
method as meeting the national surveillance
criteria for laboratory confirmation
Additional Tests: Questionable Utility
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Single-tier IgM or IgG immunoblot tests without a previous
EIA/IFA
In-house criteria for interpretation of immunoblots
Culture, immunofluorescence staining, or cell sorting of cell
wall-deficient or cystic forms of B. burgdorferi
Lymphocyte transformation tests
Quantitative CD57 lymphocyte assays
Measurements of antibodies in joint fluid (synovial fluid)
More info on www.cdc.gov/Lyme
Red Flags for Alternative Labs
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Tests offered are not FDA approved
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Laboratory claims to “specialize” in Lyme and other tickborne disease testing
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Do not accept insurance  patient pays out of pocket
($500 - $1,000 ++)
Bossuyt PM et al. Towards complete and accurate reporting of studies of
diagnostic accuracy: the STARD initiative. Fam Pract 2004;21(1):4-10.
Lyme Disease Testing:
FAQs
Does Antibiotic Prophylaxis or Treatment
Cause False Negative Serology?
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Short answer: NO
Immune response decreases when prophylaxis/treatment
has worked  infection is cleared, body has no reason to
continue producing antibodies
Thus pt may test negative, but would be a true negative at
that time
If prophylaxis fails and the patient is still infected, they will
continue to develop antibodies and should test positive on
convalescent titers
Does Immunocompromise Affect Lyme Serology?
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Short answer: UNLIKELY
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Reports of patients with significant immunocompromise
(HIV, chemotherapy for metastatic tumors, etc.) who
presented with EM
o
Produced antibodies and overall serology results similar to
immunocompetent patients
Furst et al. The impact of immunosuppression on erythema migrans. A retrospective study of clinical presentation, response to
treatment and production of Borrelia antibodies in 33 patients. Clin Exp Dermatol. 2006;31(4):509-14.
Garcia-Monco et al. Lyme disease concurrent with human immunodeficiency virus infection. Am J Med. 1989;87(3):325-8.
What To Do If I Suspect My Patient Has STARI?
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Southern tick-associated rash illness is caused by the bite of
a lone star tick. Cause is unknown.
Symptoms: EM rash, fatigue, fever, HA, myalgias, etc.
Often difficult to distinguish from Lyme disease, except by
geography or tick identification
Many physicians choose to treat with oral antibiotics
Resources for Clinicians
www.CDC.gov/lyme
www.CDC.gov/lyme --> click
on “Healthcare Professionals”
Clinician Information
http://www.cdc.gov/lyme/healthcare/clinicians.html
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Testing for Lyme Disease: Follow the Steps
PCR for Diagnosis of Lyme Disease: Is It Useful?
Southern Tick-Associated Rash Illness – When a Bull’s-Eye Rash Isn’t
Lyme Disease
CDC Resources for Clinicians & Patients
www.CDC.gov/lyme
CDC Resources for Clinicians & Patients
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We provide consultation for clinicians:
1-800-CDC-Info (general line)
970-221-6400 (DVBD Fort Collins)
[email protected]
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Additional resource: American Lyme Disease Foundation
www.aldf.com
Information for patients & clinicians
Order tick identification cards for 30¢ each
Acknowledgments
Laurie Forlano, DO, MPH
Rebecca LePrell, MPH
Virginia Department of Health
Paul Mead, MD, MPH
Anna Perea, MS
Alison Hinckley, PhD
Kiersten Kugeler, MPH
Marty Schriefer, PhD
National Center for Emerging and Zoonotic Infectious Diseases
Division of Vector-Borne Diseases l Bacterial Diseases Branch
Thank You!
Questions & Comments?
[email protected]
The findings and conclusions in this report are those of the authors and do not necessarily
represent the official position of the Centers for Disease Control and Prevention.
National Center for Emerging and Zoonotic Infectious Diseases
Division of Vector-Borne Diseases l Bacterial Diseases Branch