Understanding Laboratory Testing for Lyme Disease

Christina Nelson, MD, MPH, FAAPMedical Epidemiologist

Lyme Disease Clinician ForumJune 6th, 2013

National Center for Emerging and Zoonotic Infectious DiseasesDivision of Vector-Borne Diseases

Photo credit: CDC PHIL

Disclosures

No financial interests or relationships to disclose

Discuss: 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

Objectives

NOTE: Cases are reported based on patient's county of residence,

which may be different from where they were infected.

The challenge: B. burgdorferi cannot be easily detected from infected patients o Low # of spirochetes in blood, transient

PCR detects Bb in blood of < ½ of patients during acute dissemination (spirochetemic) phase of infection

Spirochetes can be recovered from skin biopsy specimens, but this is invasive

Therefore, indirect methods must be employed to detect infection enter serology

Background on Lyme Disease Testing

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.

Serologic Tests

1995; 44(31): 590-1

Testing is not required for patients with EM in high incidence areaso 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 diseaseo Atypical early diseaseo Bell’s palsy, arthritis, meningitis, etc.

When to Test?

Approach to Patient with Possible Early LD

Suspect Case

Consistent with EM + endemic region

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

& convalescen

t

Test acute &

convalescent

+/- treat

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 Sensitivity (%)*EM rash (acute) 38EM rash (convalescent) 67Early neurologic 87Late neurologic 100Arthritis 97

Bacon et al. JID 2003; 187:1187–99

Testing of patients with EM not generally necessary Good sensitivity in later stages of disease

*Specificity of two-tier testing is generally > 95%

Aguero-Rosenfeld, et al. Diagnosis of Lyme Borreliosis. Clin Micro Rev 2005; 18: 484-509.

IgM IgG

Positive test requires at least 2 out of 3 bands!

Note: p41 = flagellin.Present in 1/3-1/2 of all healthy people.

Positive test requires at least 5 out of 10 bands!

Interpreting the Western Blot

kD

Fla 41BmpA 39

OspC 23

1 2 43Columns: 1 = band locator 2 = intensity cutoff 3 = positive control 4 = patient sample. Band intensities below cutoff or wrong position. NEGATIVE.

False Positive IgM Western Blots

Seriburi et al. High frequency of false positive IgM immunoblots for Borrelia burgdorferi in clinical practice. Clin Microbiol Infect 2011.

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

Expanding Antibody Profiles

Aguero-Rosenfeld et al., 2005. Diagnosis of Lyme Borreliosis. Clin Microbiol. Rev. 18:484-509

IgG

1. Band locator

2. Early dissem. with neuro involvement

3. Late dissem. with arthritis

4. OspA vaccinee (31 kDa)

IgM

1.Band locator

2. Early localized with EM

3. Early dissemin. with multiple EMs

Lyme Serology Cross-Reactions

Other spirochetes• Tick-borne relapsing fever (Borrelia hermsii), syphilis

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

If signs or symptoms > 30 days, do not order IgM Western blot (second tier) test only IgG

Caution: some labs perform both IgM and IgG Western blot as default

May need to specifically request that IgM not be doneo Call lab or write instructions on order form

If not possible, at a minimum discount IgM results

Bottom Line on Serology

For patients with classic EM who live in or traveled to high incidence areas, laboratory testing is not necessaryo Clinicians may diagnose clinically and begin treatment

Two-tier serology is accurate when used appropriatelyo Sensitivity and specificity are very good in disseminated diseaseo Avoid “shotgun” testing (low pre-test probability)

Limitations: low sensitivity in early disease, cross-reaction, antibody persistence

Additional Diagnostic Tests

B. burgdorferi Culture Slow growth (days to weeks) Cultivable human samples:

EM biopsy > blood > synovial tissue > CSF Growth is detected by direct visualization

o Dark-field microscopy or fluorescent staining false-positive reads possible from thread-like cellular debris, etc.

o 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

o However, mRNA (marker of spirochete viability) not detectable

CSF positive in ~30% of pts with early neuroborreliosis Non-standardized, variety of approaches & targets

o 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

Measure CSF/serum index of antibodies to Bbo ELISA and/or Western bloto CSF and serum should be drawn simultaneously if

possible

Differential antibody expression in CSF vs. serum may occuro e.g. OspA & OspB antibodies are expressed more in CNS

PCR usually low-yield except in early neuroborreliosis

Vmp-like sequence expressed (VlsE) is an outer surface lipoprotein of Bb o C6 is a highly conserved portion of VlsE’s region 6o Highly immunogenic

C6 EIA is already FDA approved as a first-tier test Advantages over whole-cell sonicate EIA:

o Better marker of early disease o Improved sensitivity for European Lyme dzo C6 antibodies fade faster

Can parts of the two-tier approach be modified to simplify and improve accuracy in early disease?

C6

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 Developed by private laboratories Not sold or distributed across state lines

o Therefore FDA clearance not required

Clinical Laboratory Improvement Amendments (CLIA) require specific record-keeping & tests for analytic accuracyo Evaluation of clinical sensitivity and specificity not required

Alternative Tests for Lyme Disease

Several found to be unreliable

o IGenex LUAT urine antigen test 1

o Phillips culture media 2

1. Klempner, MS et al. 2001 “Intralaboratory reliability of serologic and urine testing for Lyme disease.” American Journal of Medicine 110:217-19).

2. 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

Patients and illness poorly described Nested PCR controls: B. burgdorferi, B. garinii, or B. afzelii Among the 51 samples positive by PCR:

o 40% had sequences identical to laboratory strain of B. burgdorferio > 41% identified as B. garinii or B. afzelii, strains not linked to

human illness acquired in the USo 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

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

Tests offered are not FDA approved

Laboratory claims to “specialize” in Lyme and other tick-borne disease testing

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?

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?

Short answer: UNLIKELY

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?

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

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 & Patientswww.CDC.gov/lyme

CDC Resources for Clinicians & Patients

We provide consultation for clinicians:1-800-CDC-Info (general line)970-221-6400 (DVBD Fort Collins)BZB_Public@cdc.gov

Additional resource: American Lyme Disease Foundation www.aldf.com

Information for patients & clinicians Order tick identification cards for 30¢ each

Acknowledgments

Laurie Forlano, DO, MPHRebecca LePrell, MPH

Virginia Department of HealthPaul Mead, MD, MPH

Anna Perea, MSAlison Hinckley, PhD

Kiersten Kugeler, MPHMarty Schriefer, PhD

National Center for Emerging and Zoonotic Infectious Diseases

Division of Vector-Borne Diseases l Bacterial Diseases Branch

Thank You!

Questions & Comments?

wje1@cdc.gov

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