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Evolution of Lyme Disease Testing and Future Perspectives

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Lyme disease has been a nationally recognized condition in the United States since 1991 and is the most common vector-borne illness in North America and Europe.1 Since 1991, the incidence of Lyme disease in the United States has nearly doubled and Lyme disease is now endemic to Northeastern States as well as Minnesota and Wisconsin. The Centers for Disease Control and Prevention (CDC) estimates that ~300,000 people get Lyme disease each year, however only about 35,000 cases are reported each year. This discrepancy is largely due to that fact that many cases do not get reported to the Nationally Notifiable Diseases Surveillance System (NNDSS).2 Additional limitations of surveillance data includes data are subject to each state’s abilities to capture and classify cases, and that data are captured by county of residence, not county of exposure.1 In the article below, we explore the current state of Lyme disease testing, and share perspectives on how improvements could be made in the future to improve time to diagnosis.


What is Lyme disease?

Lyme disease is a bacterial infection transmitted to humans through the bite of infected blacklegged ticks. The causative agent of Lyme disease, bacteria of the genus Borrelia, are known as spirochetes for their unique corkscrew shape.3 Several Borrelia species (spp.) have been identified and are associated with different regions including North America (Borrelia burgdorferi), Europe and Asia (Borrelia afzelii and Borrelia garinii). In the United States, B. burgdorferi is spread by deer ticks (Ixodes scapularis) in the northeastern, mid-Atlantic and north-central regions, while the western blacklegged tick (Ixodes pacificus) spread disease on the Pacific Coast.4 Recently, an additional species, B. mayonii, has been discovered in blacklegged ticks collected in northwestern Wisconsin and Minnesota and has been found to also cause Lyme disease.5

Transmission, symptoms and treatment options

Ticks often attach to hard-to-see areas such as the groin, armpits and scalp, and are difficult to identify due to their small size (<2 mm nymph stage). For Lyme disease transmission, the tick must be attached for 36 to 48 hours.4 Early symptoms of Lyme disease may begin from 3 to 30 days after the tick bite and include fever, chills, headache, fatigue, muscle and joint aches and swollen lymph nodes.6 Approximately 70–80% of people will experience the erythema migrans (EM) rash which expands gradually over time sometimes forming the classic “bulls-eye” appearance. Later symptoms, days to months after the tick bite, include headaches, facial palsy, arthritis (Lyme arthritis), joint and nerve pain, dizziness, and irregular heartbeat (Lyme carditis).6

Lyme disease can also feature neurological involvement. Neurological Lyme disease (neuroborreliosis) occurs as a secondary symptom of Lyme disease involving the peripheral or central nervous system and occurs in about 10–15% of patients with untreated Lyme disease.7 The attack on the nervous system typically appears as radiculitis (Bannwarth’s syndrome), which features cranial neuritis, facial paralysis and sensory disorders.8Less frequently, meningitis, myelitis, encephalitis and cerebral vasculitis occur.

Fortunately, patients with Lyme disease treated early with appropriate antibiotics typically experience a full recovery.9 The exact dosage and duration of treatment for Lyme disease is specific for the type of clinical manifestation. For people with EM rash, oral antibiotic treatment with either doxycycline, amoxicillin or cefuroxime is recommended.9 For patients with Lyme carditis, Lyme arthritis or neurological Lyme disease, oral or intravenous antibiotics are given.

Lyme disease diagnosis

Lyme disease diagnosis can be difficult based on symptoms alone in people who do not have an EM rash. In such cases, laboratory testing is essential.10,11 During the acute phase of the infection, molecular testing is not recommended because the DNA concentration in blood is low and may not be detectable. Later in infection, molecular testing has high sensitivity for the diagnosis of Lyme arthritis when using joint fluid as a sample (Table 1).12

Detection of antibodies in the blood is the gold standard for Lyme disease diagnosis (Table 1).12,13 The CDC established a two-tier testing algorithm in 1995, known as Standard Two-Tier Testing Algorithm (STTT). STTT consists of a first-line enzyme immunoassay (EIA) or immunofluorescence assay (IFA) screen for anti-Borrelia burgdorferi antibodies. Positive or equivocal samples are subsequently tested by B. burgdorferi-specific IgM and IgG immunoblots.13,14 Depending on the symptoms’ onset, both IgG and IgM immunoblot testing or only IgG testing have to be performed. A positive result is indicated by the presence of at least 2 out of a possible 3 bands for IgM immunoblot testing, or 5 out of a possible 10 bands for IgG immunoblot testing.15

The STTT presents some limitations: (a) In the early stage of Lyme disease, the sensitivity is low because of the low antibody concentration. As a result, false-negatives can happen. (b) Interpretation of immunoblot results can be challenging and subjective, leading to false-positive results.11,16,17 Due to these limitations, studies were performed to improve the accuracy of Lyme disease testing, and alternate testing to STTT has been additionally suggested.11,17,18 After 24 years, in July 2019, the FDA cleared a variation of STTT known as modified two-tier testing (MTTT) replacing the second tier immunoblot testing with a second EIA (either ELISA or chemiluminescence assay) that can detect IgM and IgG simultaneously or separately.19 Furthermore, the CDC and the guidelines for Lyme disease from Infectious Diseases Society of America (IDSA) updated the testing algorithm with the addition of the MTTT. 12-14

For neuroborreliosis diagnosis, either the STTT or MTTT algorithm is recommended by CDC.13 Additional testing can be performed to confirm positive results or exclude other neurological diseases (Table 1). For example, testing cerebrospinal fluid (CSF) for intrathecal antibodies or CXCL13.12,20,21

Future perspectives on Lyme disease diagnosis

MTTT's high performance has been demonstrated in various studies in the United States, Canada and Europe following FDA approval.22,23 In the United States, clinical testing labs have implemented the MTTT, and more manufacturers are offering solutions that are aligned with the MTTT. Some labs, on the other hand, continue to utilize STTT or both (MTTT and STTT) because some clinicians want to know what bands (antigens) are positive in the immunoblots to gain more information about the disease, such as those antigens that are relevant to the detection of Lyme arthritis.12 Therefore, both CDC testing algorithms are projected to be used in the coming years.

If laboratories intend to perform alternative testing in addition to, or instead of, the testing established by the CDC, they must validate the new test with the relevant assay as a lab-developed test (LDT) according to state and local guidelines. An example of alternate testing would be the use of immunoblots, as a second-tier test, containing extra and/or different antigens than those required by the CDC. A good example is the VlsE antigen, which stands for variable major protein-like sequence expressed. According to studies, adding the VlsE antigen, in an immunoblot to detect IgG, improves the sensitivity of Lyme disease testing in the early and late stages while retaining a high level of specificity.16,24-26

Patients who have recently traveled to Europe or Asia may benefit from testing for European or Asian Borrelia species in addition to US species. Due to the similarity of antigenic epitopes of Borrelia species antibody cross-reactivity is observed within the Borrelia burgdorferi sensu lato complex.27,28 As there may be species-specific antibodies in some circumstances, adding particular antigens for each species can improve the sensitivity of the tests.29

New assays are being investigated due to the limitations of serology during the early stages of Lyme disease. For example, new technologies with high sensitivity are being developed in the field of molecular biology.30,31 In serology, the discovery of novel antigens is crucial. According to a recent study, detecting antiphospholipid antibodies can improve STTT sensitivity during the early stages of illness.32 Additionally, T-cell response detection against Borrelia spp. infection has been explored, implying that T-cell response testing could aid in the diagnosis of early Lyme disease.33-36

Further studies are required to support the inclusion of such testing alternatives in the CDC testing algorithm and to better understand the clinical utility of new antigens and markers for the early phase of the infection and their relevance to monitoring treatment and reinfection.

Table 1: Clinical symptoms and testing in early and convalescent phases of Lyme disease. 


Lyme disease stage

Clinical symptoms/testing

Early stage of Lyme disease                                        (3-30 days after tick bite)

Disseminated and chronic stages of Lyme disease

(>30 days after tick bite)

Clinical symptoms

EM rash: detectable in 70%–80 % of patients

Unspecific: fever, chills, headache, fatigue, muscle, and joint aches, swollen lymph nodes

EM rash: not observed

Lyme arthritis: swollen knees, neck stiffness

Lyme carditis: light-headedness, fainting, shortness of breath, heart palpitations, or chest pain

Neuroborreliosis: meningitis/encephalitis


PCR (limited utility in blood)

Lyme arthritis: PCR                                                 (highest sensitivity in joint fluid)


·   IgM

·   Antibodies against phospholipids*

IgM and/or IgG are detectable in the blood depending on the time of the infection


Neuroborreliosis (CSF testing):

·         Intrathecal IgM and/or IgG detectable depending on time of the infection

·         CXCL13 upregulation

Cellular response

T-cell response*

T-cell response*

* Further studies are required to support the clinical utility of this testing.

About the authors:

Maite Sabalza, PhD, is the Scientific Affairs Manager at EUROIMMUN US, a PerkinElmer company. Her academic background is in infectious diseases and diagnostics.

Ilana Heckler, PhD, is the Scientific Affairs Associate at EUROIMMUN US, a PerkinElmer company. She holds a PhD in Chemical Biology for her studies on bacterial hemoprotein sensors of nitric oxide.


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