Lyme Disease – Serology

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This page provides routine serology (antibody) testing information for Lyme disease (or Lyme Borreliosis) at Public Health Ontario’s (PHO) laboratory as well as testing referred out to the National Microbiology Laboratory (NML). For information regarding other testing options, refer to Lyme Disease PCR page.

Lyme disease is caused by spirochetal bacteria of the genus Borrelia. In North America, the main causative agent is Borrelia burgdorferi sensu stricto, while in Europe, B. burgdorferi, B. afzelii and B. garinii are the relevant genospecies. In Russia and Northern Asia, B. garinii is the most common species causing Lyme disease. As a vector borne zoonotic disease, the bacterium is transmitted through the bite of a species of Ixodes ticks, thus most cases occur during the warmer months when the ticks are active.

Initial symptoms may vary from asymptomatic to a combination of fever, headache, myalgia, stiff neck and fatigue, and may be accompanied by a characteristic circular or bull’s eye rash called erythema migrans (EM). Untreated, the disease may progress to the joints, the heart, and the nervous system.

Updates:

As of April 1st 2023, PHO’s testing for B. burgdorferi antibodies uses a modified two-tiered testing approach (MTTT). See Test Method(s) section for details.

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Testing Indications

Serological testing of asymptomatic patients, including individuals following a tick bite, is not recommended.
Serological testing for individuals with Erythema migrans, acute phase (seasonal occurrence and exposure in an endemic/risk area) is generally not recommended.
Serological testing is useful for individuals in the absence of a rash, presenting with non-specific symptoms where Lyme disease is suspected. An acute and convalescent specimen (2-4 weeks apart) may be required for laboratory confirmation of Lyme disease.

Acceptance/Rejection Criteria

Serological tests should not be used to assess treatment response, as antibodies persist long after treatment. Specimens submitted for test of cure will be cancelled.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
Borrelia burgdorferi or Lyme disease^1,2	General Test Requisition	Whole blood Serum	5.0 ml whole blood or 1.0 ml serum	Clotted whole blood or serum separator tubes (SST) with the gel sediment
Borrelia spp. CSF Serology^1,2,3	General Test Requisition	Requires fresh serum and CSF³	0.5 mL CSF AND 0.5 ml serum	Collect blood in SSTs. Collect CSF into sterile tube without preservatives
European Lyme^1,2,4 B. garinii B. afzelii	General Test Requisition	Whole blood Serum	5.0 ml whole blood or 0.25 ml serum	Clotted whole blood or SST

Submission and Collection Notes

Complete all fields of the PHO General Test Requisition Form. Include the patient’s full name, date of birth, and Health Card Number (must match the specimen label), physician name and address, and relevant clinical information:

Enter Borrelia burgdorferi or Lyme disease under ‘Test description’.
Date of symptom onset
Date of collection
Clinical signs and symptoms
Recent Travel history (within 12 months)
Exposure history (e.g. tick bite)

Note: Specimens may be delayed or rejected if they are not the appropriate specimen type, have insufficient volume, or are not accompanied by relevant patient information.

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.

Borrelia spp. CSF serology (IgG EIA) requests require prior approval. Prior to submission, notify PHO’s laboratory Customer Service Centre at 416-235-6556/ 1-877-604-4567 to obtain approval from a PHO laboratory microbiologist.

Paired serum and CSF specimens should be collected from the patient within one-hour of each other, up to a maximum of 24 hours. If this timeframe is exceeded, the test results may be invalid. Additional mandatory information for testing is required:

Exposure history to blacklegged ticks accompanied by clinical signs and symptoms of neuroborreliosis, and
Absolute values for total albumin and total IgG concentrations of both the serum and CSF specimens in order to perform interpretive calculations for this test.
Specimens are referred out to NML for testing.*

When European Lyme is suspected, European Lyme serology should be specifically stated on the requisition along with exposure to tick bite and RECENT travel history (≤ 12 months) including location and dates. Specimens may be delayed or rejected if they do not include this information.

Specimens are referred out to NML* for testing if PHO’s European Lyme EIA screen result is indeterminate or reactive.

*Refer to the NML website: Pathogens - B - Guide to Services - CNPHI (canada.ca) for testing information.

Timing of Specimen Collection

Acute (collected early after the onset of symptoms) and convalescent (collected 2-4 weeks after the onset of symptoms) specimens may be required for the confirmation of laboratory based diagnosis.

Limitations

Haemolysed, icteric, lipemic or microbial contaminated sera or plasma are not recommended for testing.

Storage and Transport

Centrifuge if using SST. Place specimen in biohazard bag and seal. Specimens should be stored at 2-8°C following collection and shipped to PHO’s laboratory on ice packs.
All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

CSF specimens for serological testing may be refrigerated up to 5 days following collection and shipped to PHO’s laboratory ASAP on ice packs.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

Lyme MTTT serology is performed daily at PHO’s laboratory during peak season (summer – fall), and performed 2-3 times per week during off season (winter – spring).

Testing performed at PHO’s laboratory has a turnaround time (TAT) of 5 days or less for non‐reactive results and up to 7 days for reactive results from receipt at PHO’s laboratory.

Testing performed at NML has a TAT of up to 21 days from receipt at NML.

STAT testing is not available.

Specimen testing may be expedited in unique circumstances. Please contact PHO’s laboratory Customer Service at 416-235-6556 or 1-877-604-4567.

Test Methods

Serology is the main laboratory diagnostic tool available for Lyme disease.

As of April 1st 2023, PHO’s laboratory offers MTTT Borrelia burgdorferiELISA testing. Initial screening (Tier 1) is done by IgG/IgM enzyme-linked immunosorbent assay (ELISA) using a whole cell lysate. Tier 1 reactive or indeterminate specimens are further tested (Tier 2) on a second IgG/IgM ELISA targeting specifically VlsE1 and pepC10 antigens. Immuno Blot assay for Lyme serology is no longer being offered at PHO’s laboratory.

Test Limitations:

Serological testing is known to be less sensitive in early onset of Lyme disease presentation (i.e. early localized disease). Therefore, negative serological testing results do not rule out Lyme disease. The sensitivity of serological testing increases significantly for patients presenting with early disseminated and late disseminated Lyme disease.
Serological testing cannot differentiate between past infections and new infection as antibodies persist for years.
Serological testing should not be used to assess treatment response, as antibodies persist after treatment.

Testing performed at NML includes:

European Lyme testing (If PHO’s European Lyme EIA screen is indeterminate or reactive)
PCR testing for B. burgdorferi and Borrelia spp on CSF, synovial fluid, tissue etc.
CSF serology (IgG EIA) - with restrictions

Algorithm

PHO’s laboratory performs tests for antibodies to B. burgdorferi using a modified two-tier test algorithm (MTTT). This algorithm is endorsed by both the Center for Disease Control (CDC) as well as the Canadian Public Health Laboratory Network (CPHLN) as an acceptable alternative method for detection of antibodies against B. burgdorferi.

MTTT algorithm does not test for European Lyme disease. If European Lyme disease is suspected, please submit a request for European Lyme disease along with appropriate travel history. PHO will use the Zeus Borrelia VlsE1/pepC10 IgG/IgM test system to screen for European Lyme. Specimens are forwarded to NML for serology confirmation only if the screen result is indeterminate or reactive. Non-Reactive specimens will not be sent to NML as PHO laboratory’s ELISA test reliably detects antibodies to European genospecies of Borrelia.

Interpretation

The following table provides possible test results with associated interpretations:

Lyme Tier 1 EIA	Lyme Tier 2 EIA	Borrelia burgdorferi (Lyme disease) serology interpretation
Non- Reactive	Not tested	Non- Reactive Advise follow-up specimen (2-4 weeks apart) if clinically indicated.
Indeterminate	Non- Reactive	Non- Reactive The results should be interpreted within the clinical context. Advise follow-up specimen (2-4 weeks apart) if clinically indicated.
Indeterminate	Indeterminate	Indeterminate The results should be interpreted within the clinical context. Advise follow-up specimen (2-4 weeks apart) if clinically indicated. Copy of results sent to MOH.
Indeterminate	Reactive	Reactive The results should be interpreted within the clinical context. Copy of results sent to MOH.
Reactive	Non- Reactive	Non- Reactive The results should be interpreted within the clinical context. Advise follow-up specimen (2-4 weeks apart) if clinically indicated.
Reactive	Indeterminate	Reactive The results should be interpreted within the clinical context. Copy of results sent to MOH.
Reactive	Reactive	Reactive The results should be interpreted within the clinical context. Copy of results sent to MOH.

Reporting

Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens that are reactive for Borrelia species antibodies are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

NML results are received back at PHO’s laboratory, and reports are forwarded to the ordering physician or health care provider indicated on the requisition.

Data and Analysis

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Mis à jour le 28 mars 2023