IGeneX Reference Laboratory

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Links Between Testing and Reporting from the
Laboratory Perspective Jyotsna Shah,
Ph.D, CMLD, MBA February 25, 2004
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IGeneX Reference Laboratory
Specializes in Diagnosis of Lyme and Other
Tick Borne Diseases
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Other Tick-borne Diseases Associated With Lyme
Disease

• Ehrlichiosis
• Human Granulocytic (HGE)
• Human Monocytic (HME)
• Babesiosis
• B. wa-1
• B. microti?
• Bartonella
• B. henselae

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Lyme Disease Testing
Indirect Tests Detection of Patients 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
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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 46RR10
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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

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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)

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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.
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
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• Engstrom SM, E. Shoop, and R. Johnson. 1995.
  Immunoblot Interpretation for Serodiagnosis of
  Early Lyme Dis. J. Clin. Micro. 33419-427.
• 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

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

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HGE Human Granulocytic Ehrlichia HME Human
Monocytic Ehrlichia Titer of gt1160 is considered
positive