1. Understanding Laboratory Testing for Lyme Disease Christina Nelson, MD, MPH, FAAP Medical Epidemiologist Lyme Disease Clinician Forum June 6 th, 2013 National Center for Emerging and Zoonotic Infectious Diseases Division of Vector-Borne Diseases Photo credit: CDC PHIL

2. Disclosures  No financial interests or relationships to disclose

3. 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

4. NOTE: Cases are reported based on patient's county of residence, which may be different from where they were infected.

5.  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

6.  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

7. 1995; 44(31): 590-1

8.  Testing is not required for patients with EM in high incidence areas o 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 Atypical early disease o Bell’s palsy, arthritis, meningitis, etc. When to Test?

9. 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 & convalescent Test acute & convalescent +/- treat Treat Adapted from: Goldsmith et al. Fitzpatrick’s Dermatology in General Medicine, 8 th edition.

10. Nuts & Bolts of Serologic Testing for Lyme Disease

12. Sensitivity of Two-Tier Serologic Testing Lyme Disease StageSensitivity (%)* EM rash (acute)38 EM rash (convalescent)67 Early neurologic87 Late neurologic100 Arthritis97 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%

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

14. kD Fla 41 BmpA 39 OspC 23 12 4 3 Columns: 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

15. Expanding Antibody Profiles Aguero-Rosenfeld et al., 2005. Diagnosis of Lyme Borreliosis. Clin Microbiol. Rev. 18:484-509 IgG 1. B and locator 2.Early dissem. with neuro involvement 3.Late dissem. with arthritis 4.OspA vaccinee (31 kDa) IgM 1. B and locator 2.Early localized with EM 3.Early dissemin. with multiple EMs

16. 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

17. Take-Home Points: Appropriate Use of Testing

19. Haiku to Lyme Disease Testing Where disease is rare Positives mostly deceive Even with good tests

20. 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 done o Call lab or write instructions on order form  If not possible, at a minimum discount IgM results

21. Bottom Line on Serology  For patients with classic EM who live in or traveled to high incidence areas, laboratory testing is not necessary o Clinicians may diagnose clinically and begin treatment  Two-tier serology is accurate when used appropriately o Sensitivity and specificity are very good in disseminated disease o Avoid “shotgun” testing (low pre-test probability)  Limitations: low sensitivity in early disease, cross-reaction, antibody persistence

22. Additional Diagnostic Tests

23. 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.

24. 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.

25. CSF Testing for Neuroborreliosis  Measure CSF/serum index of antibodies to Bb o ELISA and/or Western blot o CSF and serum should be drawn simultaneously if possible  Differential antibody expression in CSF vs. serum may occur o e.g. OspA & OspB antibodies are expressed more in CNS  PCR usually low-yield except in early neuroborreliosis

26.  Vmp-like sequence expressed (VlsE) is an outer surface lipoprotein of Bb o C6 is a highly conserved portion of VlsE’s region 6 o 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 dz o C6 antibodies fade faster  Can parts of the two-tier approach be modified to simplify and improve accuracy in early disease? C6

27. C6 As Second Tier? Standard 2-tier2-EIAC6 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

28. Alternative Tests for Lyme Disease

29. “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 accuracy o Evaluation of clinical sensitivity and specificity not required

30. 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).

31. Alternative “Culture” Test

32. 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. burgdorferi o > 41% identified as B. garinii or B. afzelii, strains not linked to human illness acquired in the US o All three genospecies were used as controls in the nested PCR reactions

33. 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

34. 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

35. 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 ++)

36. Bossuyt PM et al. Towards complete and accurate reporting of studies of diagnostic accuracy: the STARD initiative. Fam Pract 2004;21(1):4-10.

37. Lyme Disease Testing: FAQs

38. 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

39. 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.

40. 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

41. Resources for Clinicians

42. www.CDC.gov/lyme

43. www.CDC.gov/lyme --> click on “Healthcare Professionals”

44. 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

45. CDC Resources for Clinicians & Patients www.CDC.gov/lyme

48. 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

49. 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

50. 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