1. Links Between Testing and Reporting from the Laboratory Perspective Jyotsna Shah, Ph.D, CMLD, MBA February 25, 2004

2. IGeneX Reference Laboratory Specializes in Diagnosis of Lyme and Other Tick Borne Diseases

3. Other Tick-borne Diseases Associated With Lyme Disease Ehrlichiosis Human Granulocytic (HGE) Human Monocytic (HME) Babesiosis B. wa-1 B. microti? Bartonella B. henselae

4. Lyme Disease Testing Indirect Tests: Detection of Patient’s Immune responses to Borrelia burgdorferi, causative agent of Lyme Disease. Serology (C6), Western blots, Immunofluorescence Direct Tests: Detection of the Borrelia burgdorferi specific proteins (antigens), DNA and RNA, in patient clinical specimen (blood, serum, urine, CSF, etc.) Lyme Urine Antigen, PCR, Reverse Western blot

5. Lyme Disease Case Classification by CDC CONFIRMED CASE A Case with EM, or A Case of Late Manifestation that is Laboratory Confirmed. Laboratory Confirmation Isolation of Borrelia burgdorferi from a clinical sample or Demonstration of IgM or IgG antibodies to B. burgdorferii in serum or CSF. A two-test approach using a sensitive ELISA or IFA, followed by Western Blots. NOTE: The above is a SURVEILLANCE case definition, developed for national reporting of Lyme Disease by CDC. IT IS NOT INTENDED FOR USE IN CLINICAL DIAGNOSIS. Ref: MMWR 46:RR10

7. IgG WESTERN BLOT IGeneX Reference Laboratory 23-25 kDa (Osp C) 31 kDa (Osp A) 34 kDa (Osp B) 39 kDa 41 kDa (Flagella) 93 kDa (83 kDa?) CDC/ASPHLD 18 kDa 23-25 kDa (Osp C) 28, 30 kDa 39 kDa 41 kDa (Flagella) 45, 58, and 66 kDa 93 kDa

8. IgM WESTERN BLOT IGeneX Reference Laboratory 23-25 kDa (Osp C) 31 kDa (Osp A) 34 kDa (Osp B) 39 kDa 41 kDa (Flagella ) CDC/ASPHLD 23-25 kDa (Osp C) 39 kDa 41 kDa (Flagella)

9. REFERENCE Bakker LL, Callister SM, Wantol PG and Shell RF. Interlaboratory comparison of test results of detecting Lyme disease by 516 participants in the Wisconsin State Laboratory of hygiene College of American Pathologists proficiency testing Program. J. Clin Microbiol. 1997 537-543. ILADS In fact, studies conducted by the group responsible for Lyme Disease proficiency testing for the College of American Pathologists (CAP) concluded that the currently available ELISA assays for Lyme Disease do not have adequate sensitivity to be part of the two-tiered approach of the CDC/ASHLD, whereby only ElISA-positive samples can be tested by Western blotting.

10. Engstrom SM, E. Shoop, and R. Johnson. 1995. Immunoblot Interpretation for Serodiagnosis of Early Lyme Dis. J. Clin. Micro. 33:419-427. 55 patients with EM Blood Collected on 5 visits over 4 months Patients were positive on one or more visits 20% of patients remained seronegative In control group that might resemble Lyme IgM and IgG, WB specificities were 93-94% 2/5 IgG bands (20, 23-25, 35, 39, 93) scored

12. Presence of Nucleic Acid As Confirmed Diagnosis For the diseases listed below, CDC considers, the presence of infectious agent’s Nucleic Acid in a clinical sample, acceptable as confirmed diagnosis of the disease. (MMWR 46:RR-10) Bacteria (Nucleic Acid Amplification) Chlamydia Gonorrhea Pertussis Viruses Hantavirus Pulmonary Syndrome (PCR) Arboviral Encephalitis (Genomic Sequences) Why Not For LYME DISEASE?

15. HGE = Human Granulocytic Ehrlichia; HME = Human Monocytic Ehrlichia Titer of >1:160 is considered positive