Lyme disease: sample testing advice
Information to assist with the laboratory diagnosis of Lyme disease and requirements for sample submission.
Access to Lyme disease testing services
This guidance on the laboratory diagnosis of Lyme disease is intended for healthcare professionals in the UK. Patients concerned about possible Lyme infection should consult an appropriate healthcare professional, for example their GP, in the first instance.
Health professionals wishing to discuss a possible case or ascertain local arrangements for testing should contact a local Infection specialist (such as a consultant in infectious diseases or microbiology).
NHS testing for Lyme disease is available through local service providers and the Rare and Imported Pathogens Laboratory (RIPL) at UK Health Security Agency (UKHSA) Porton where ISO15189 accredited confirmatory testing is also provided. RIPL also provides a testing service for neurological Lyme disease.
RIPL provides medical and laboratory specialist services to the NHS and other healthcare providers, covering advice and diagnosis of a wide range of unusual bacterial and viral infections, including Lyme disease.
RIPL continuously updates its methods and will make further information on Lyme disease diagnostic testing available as it arises.
Management of individuals without symptoms following a tick bite
Diagnostic testing is not recommended for individuals who do not develop any symptoms suggestive of Lyme disease after a tick bite.
Some commercial companies offer services to test removed ticks for the presence of the bacteria that cause Lyme disease. UKHSA does not provide such tick-testing services. The results of such tests should not be used to inform diagnosis or treatment. A positive result does not mean that the infected tick will have passed on the bacteria – there are many factors that determine whether Lyme disease results from the bite of an infected tick. A negative result may not be technically valid and could give false assurance, as it does not exclude the possibility that another tick elsewhere on the body has been missed by the patient.
UKHSA runs a tick surveillance scheme and is happy to receive ticks for species identification and to monitor tick distribution.
Investigation of suspected Lyme disease
Erythema migrans is a clinical diagnosis and does not require confirmation by laboratory testing. Lyme disease is not a notifiable disease so there is no statutory requirement to notify clinically suspected cases to the local Health Protection Team.
See additional information about differential diagnoses for erythema migrans and other Lyme disease–like symptoms.
The 2018 NICE Lyme disease guideline provides detailed advice about when a diagnosis of Lyme disease should be suspected and about which tests to use and when.
The NICE Lyme disease guideline also contains a useful summary diagram of the routine serological testing recommendations for Lyme disease.
Investigation of suspected neurological Lyme disease
The diagnosis of neurological Lyme disease can only be confirmed by examination of the CSF and a paired serum sample. A definite diagnosis is based on the presence of a pleocytosis (usually predominantly lymphocytes) in the CSF, demonstration of intrathecal synthesis of specific antibodies to Borrelia species in CSF by comparison to serum and the presence of neurological symptoms. It is not possible to confirm intrathecal synthesis and hence definite neurological Lyme disease without a paired serum (see European Federation of Neurological Societies guidance).
In 2018 RIPL introduced a service for the detection of intrathecal synthesis of Borrelia-specific antibodies which is summarised in this flow diagram. An accessible text version of this flowchart is available below.
Clinicians may ask for guidance on laboratory testing of CSF from RIPL if required.
Sample types for Lyme disease testing
For routine Lyme disease serological testing, you must send a serum sample (>0.5 mL).
For testing for neurological Lyme disease, you must send paired CSF (>0.5 mL) and serum (>0.5 mL) samples taken on the same day. If possible, please provide CSF cell count and total albumin, IgG and IgM values. If albumin, IgG and IgM measurements are not available, RIPL will make arrangements for these measurements at additional cost.
PCR testing is available. We recommend that specialists call RIPL to discuss appropriate sample types for PCR. These may include joint fluid, biopsy tissue, CSF and EDTA plasma. Please submit biopsies as fresh tissue in a sterile container, ideally with a drop of sterile saline to prevent the tissue drying out.
Completion of Lyme disease test request form (P2)
Diagnostic samples must be submitted with a completed RIPL Lyme disease test request form (P2). Please use a single request form for each patient even when sending several samples.
Please provide details of the sample type(s), patient’s symptoms and onset date, tick bite or exposure history and, if relevant, travel and treatment history. Symptom onset date is particularly important for interpretation of laboratory results.
When sending CSF, please provide the cell count and total albumin, IgG and IgM levels for the CSF and serum pair, if available.
Ideally the clinician who sees the patient should print out and complete the appropriate request form (except for the sender’s information at the top). Send the request to the local laboratory with the clinical sample(s) along with a local laboratory request form, whether this is paper or electronic.
The local laboratory should complete the sender’s information on the request form and then forward the completed form and sample(s) to RIPL. Before sending samples, clinicians are advised to liaise with their local laboratory because local arrangements may vary.
If only immunoblot confirmation is required because the local laboratory has already obtained a positive Lyme screening test result on the serum sample being submitted, tick the “Line blot confirmation only” box and write the positive screening test result below.
Lyme disease laboratory tests available at RIPL
The primary service provided by RIPL is serological testing using well-characterised and validated screening and confirmatory tests in accordance with the NICE Lyme disease guideline.
RIPL participates in regular external quality assurance exercises as an independent measure of its performance.
Details of prices and turnaround times for Lyme testing are provided in Appendix 1 of the RIPL user manual.
Serological testing of serum for the diagnosis of Lyme disease
The most commonly used tests look for antibodies to the Borrelia species that cause Lyme disease in the UK and Europe, but they also detect infections from strains of Borrelia from the US.
The antibody response takes several weeks to reach a detectable level, so antibody tests in the first few weeks of infection may be negative. If the first sample was taken within 4 weeks from the onset of symptoms and is negative and there is a clinical suspicion of Lyme disease, then retesting in 4 to 6 weeks may be useful.
It is very rare for patients to have negative antibody tests in longstanding infections. Borrelia antibodies persist indefinitely in some patients and this does not indicate continuing disease or a need for re-treatment.
Serological testing for Lyme disease in the UK and much of the world follows a two-step approach:
- The first stage of testing uses a sensitive screening ELISA test. Since 17 Dec 2020, RIPL has used the Borrelia VlsE1/pepC10 IgG/IgM ELISA Test System (Zeus Scientific). This replaced the C6 Lyme ELISA (Immunetics) which was withdrawn from the market for commercial reasons.
- Sensitive tests have the disadvantage of occasionally detecting other diseases and producing false positive results, so a second more specific confirmatory test is run on all samples giving a positive or indeterminate preliminary screening test result. RIPL uses the Borrelia ViraChip® IgG, IgM test (Viramed Biotech AG) to confirm the presence of Borrelia-specific antibodies .
Serological testing of CSF for the diagnosis of neurological Lyme disease
Serological testing for neurological Lyme disease is based on demonstrating intrathecal synthesis of Borrelia-specific antibodies in CSF. For laboratory testing for neurological Lyme disease, IgG ViraChip® serology assays are performed on CSF and paired serum and the results compared.
CSF samples must be tested in parallel with a contemporaneous serum sample and albumin and total IgG levels compared between the 2 sample types to produce a meaningful result.
For necessary sample types and volumes see Sample types for Lyme disease testing.
Molecular testing for detection of Borrelia species bacterial DNA
PCR is available for Borrelia species DNA detection but is of limited value in routine testing for Lyme disease because the organism is only present in blood during the early stages of the disease and is predominantly restricted to the affected tissues.
Diagnostic molecular testing for Borrelia species DNA is available on request for relevant specimen types. Please call RIPL to discuss individual cases.
Alternative diagnostic tests not available at RIPL
Tests used by the NHS and UKHSA to identify Lyme disease are well characterised, standardised, and are highly reproducible between laboratories. They are the methods of choice recommended in the 2018 NICE Lyme disease guidelines, following an extensive review of the evidence and literature. International external quality assurance (EQA) schemes are in place to ensure consistency between different centres offering these tests.
Several private laboratories in Europe and the US offer an alternative type of test called an ELISpot to diagnose Lyme disease. This looks for different markers in blood samples compared to conventional validated Lyme disease serology tests. The laboratories using these tests in the diagnosis of Lyme disease do not publish their methods, and have not produced peer reviewed publications on their clinical value.
This makes it very difficult to verify their results, especially as there are no national or international EQA schemes for Lyme disease ELISpot tests and therefore no independent verification of performance between laboratories. Without independent evidence it is impossible to determine the validity of results produced using these alternative tests.
Other diagnostics tests such as CD57 test and direct microscopy are also advertised online. Like ELISpot tests, these assays are not recommended since robust clinical studies demonstrating their specificity and sensitivity for the diagnosis of Lyme disease do not exist. Similarly, independent external quality assurance schemes are not available for these tests.
RIPL cannot interpret the results of alternative diagnostic tests.
Interpretation of Lyme disease results from RIPL
Negative ELISA on serum
Early clinical Lyme disease in the form of erythema migrans with an associated history of a tick bite should be treated empirically. There is no need for testing unless there are further symptoms.
A negative ELISA result in the early stages of Lyme disease (within 4 weeks of symptom onset) does not exclude infection. If acute Lyme disease is suspected but serology results are negative, we recommend that the test is repeated in 4 to 6 weeks with a fresh sample to look for seroconversion.
In patients with long term symptoms a negative ELISA test usually excludes Lyme disease as a cause of these symptoms. Information on differential diagnosis for patients with persistent symptoms and negative Lyme disease serology results is available.
Positive ELISA on serum
RIPL will automatically proceed to do IgM and IgG immunoblot tests after a positive or indeterminate ELISA test and will provide an overall interpretation of the ELISA and immunoblot in the light of the clinical details provided on the request form.
Please provide clinical details to allow the interpretation of serological results. These are needed for interpretation because borrelia-specific antibodies may persist for several years in patients who have had Lyme disease in the past, long after the bacteria have been cleared from the body. Therefore, detection of borrelia specific antibodies in someone with no evidence of current clinical symptoms or recent tick exposure argues against active Lyme disease infection. After successful treatment of Lyme disease antibody concentrations may slowly fall over time.
Borrelia species are notifiable organisms. The numbers of positive results from laboratory confirmed cases in RIPL are reported to UKHSA and analysed for inclusion in UKHSA Health Protection Reports as part of Lyme disease epidemiology and surveillance.
CSF serology results
A positive CSF serology result to any Borrelia antigen that is not present in paired serum, or a higher blot intensity in CSF serology compared to paired normalised serum is evidence of intrathecal synthesis of Borrelia-specific antibodies and supports the diagnosis of neurological Lyme disease. The ViraChip analyser software automatically determines whether a particular serology result is positive or negative and no additional interpretation is permitted.
Note that:
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a negative serum antibody result does not exclude a positive CSF result in early neurological Lyme disease, especially in children
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neurological Lyme disease may not give rise to detectable antibodies in CSF so a negative CSF blot result does not exclude neurological Lyme disease as a cause of e.g. facial palsy in a child
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reactivity to the borrelial OspC antigen in CSF has been associated with cross-reacting EBV antibodies in some individuals, especially in children - if this is the only evidence of neurological Lyme disease, the differential diagnosis should be considered
As with serum serology, CSF test results are interpreted in the light of the clinical information provided.
If Borrelia-specific antibodies are not detected in paired serum and CSF samples collected 12 or more weeks after the onset of symptoms, then Lyme disease is highly unlikely.
Borrelia species PCR results
Borrelia species DNA may occasionally be detected in the blood by PCR, but a negative PCR test is of no value in excluding localised Lyme disease.
The overall sensitivity of PCR on a skin biopsy of an EM or ACA rash is around 50% and is limited by the chance of a single biopsy hitting a site with a significant number of organisms.
In neurological Lyme disease involving the CNS, up to 10% of cases may be PCR positive on a CSF sample; a negative PCR result does not exclude the diagnosis.
Synovial fluid may be positive by PCR in up to 50% of cases. A negative result does not exclude the diagnosis.
Contact information
Lyme disease diagnostic service
Rare and Imported Pathogens Laboratory (RIPL)
UKHSA, Manor Farm Road
Porton Down
Wiltshire
SP4 0JG
Email [email protected]
Telephone 01980 612348 (available 9am to 5pm, Monday to Friday)
DX address DX 6930400, Salisbury 92 SP
Neurological Lyme disease: laboratory investigations and diagnosis
Accessible text version of the flowchart
If neurological Lyme disease is suspected by a specialist doctor:
- offer a test for intrathecal antibody production
- take a paired serum and CSF sample (each greater than or equal to 500 µL)
Send the paired serum and CSF sample to the UKHSA Lyme disease service along with (if known):
- the CSF cell count
- the serum total albumin and IgG
- the CSF total albumin and IgG
If not known, the UKHSA Lyme disease service will measure these last 2 parameters.
The UKHSA Lyme service will carry out an IgG immunoblot test on the CSF and appropriately diluted paired serum.
If there is a positive IgG immunoblot result, this is indicative of intrathecal production of antibodies to Borrelia spp. If the symptoms are compatible with neurological Lyme disease and/or there is evidence of pleocytosis in the CSF, treat.
If there is no positive IgG immunoblot result, offer a polymerase chain reaction (PCR) test on the CSF if symptoms are less than or equal to 6 weeks in duration and there is enough CSF.
If the PCR result is positive it means Borrelia spp DNA has been detected in the CSF, which is diagnostic of neurological Lyme disease, and the patient must be treated.
If the PCR result is negative, consider an alternative diagnosis. If Lyme remains a possibility, offer a screening ELISA test on serum in 6 weeks.
Updates to this page
Published 31 July 2018Last updated 20 April 2022 + show all updates
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Updated Serological testing of serum for the diagnosis of Lyme disease section and added text version of laboratory investigation flowchart.
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First published.