The diagnosis of borreliosis often begins with a detailed clinical evaluation. A history of exposure to ticks, the presence of characteristic signs such as EM, and symptoms such as fatigue, fever, headache, or musculoskeletal pain are key elements considered by clinicians. However, clinicians should be aware of all possible symptoms that have not found a place in the official traditional literature.
Although clinical diagnosis plays an important role, it can be limited due to the nonspecific nature of symptoms that may overlap with other conditions.
Serological Testing: Enzyme-Linked Immunosorbent Assay (ELISA)
The cornerstone of traditional laboratory diagnosis of Lyme disease is serology, specifically the detection of antibodies against Borrelia burgdorferi using the enzyme-linked immunosorbent assay (ELISA). ELISA is a first-line screening tool designed to detect immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies produced in response to Borrelia infection.
Limitations
ELISA has several limitations. It has a high false-negative rate in early disease because antibodies may not yet be present in detectable levels during the acute phase. Additionally, false positives can occur due to cross-reactivity with other infections such as syphilis, Epstein-Barr virus, or autoimmune disorders.
There have also been numerous confirmed cases of false negatives, along with reports of critically ill patients who received multiple negative ELISA results before finally obtaining conclusive proof of infection.
Confirmatory Testing: Western Blot
Following a positive or equivocal ELISA result, the diagnosis is typically confirmed with Western blotting, which detects specific Borrelia proteins in a more detailed and specific manner.
Western blot separates proteins by molecular weight, enabling the detection of antibodies against specific Borrelia antigens. This method distinguishes between IgM and IgG antibodies, which is traditionally thought to help determine the stage of infection. However, there have been cases of long-term infected patients who remained untreated due to a missed diagnosis, showing only the presence of IgM antibodies.
CDC Guidelines
The U.S. Centers for Disease Control and Prevention (CDC) recommends a two-step process, where a positive or indeterminate ELISA result is followed by a Western blot test. A positive diagnosis requires the presence of at least 2 out of 3 IgM bands in early-stage disease or 5 out of 10 IgG bands in late-stage disease.
Limitations
Like ELISA, Western blotting is not without issues. It is more technically demanding, requires interpretation by experienced laboratory personnel. False negatives can occur in the early stages of infection when antibody production is low.
As with ELISA cases, there have been instances where patients received repeated negative Western blot results, only to have the infection detected through subsequent tests.
Polymerase Chain Reaction (PCR) Testing
PCR testing detects Borrelia DNA in biological samples such as blood, cerebrospinal fluid (CSF), or synovial fluid. PCR is more sensitive than serology and can identify the presence of Borrelia even in the absence of an immune response.
PCR is particularly useful in diagnosing later stages of Borreliosis, such as Lyme arthritis, where the bacterial load may still be detectable. It is also beneficial in cases of neuroborreliosis, where Borrelia DNA may be present in the CSF.
Limitations
The major limitation of PCR is that Borrelia spirochetes are often present in low numbers in body fluids, especially during early infection. Therefore, a negative PCR result does not rule out infection. Furthermore, the test is highly dependent on the quality of the sample and may require invasive procedures, such as lumbar puncture, to obtain appropriate specimens.
Emerging Diagnostic Methods
Serologic Advances: C6 Peptide ELISA
A more recent advance in serological testing for Lyme disease is the use of C6 peptide ELISA. The C6 peptide is a conserved region of the Borrelia outer surface protein (OspC), which elicits a strong immune response. This test can detect both early and late-stage Lyme disease, offering improved sensitivity over traditional ELISA.
Advantages
The C6 peptide assay has the advantage of detecting infection earlier than conventional ELISA and is less prone to cross-reactivity with other diseases. Furthermore, it provides a higher degree of specificity for active Lyme disease.
Limitations
While it has shown promise, C6 peptide ELISA is not universally available and has limitations similar to other serologic tests, such as inability to detect infection in the very early stages before antibody production begins.
T-Cell Based Assays
Recent developments in immunodiagnostics have led to the creation of T-cell-based assays, which aim to detect Borrelia-specific cellular immune responses rather than antibodies. These tests, such as the ELISpot assay, can detect T-cells that have been activated by Borrelia antigens, potentially identifying infection earlier than serology-based methods.
Advantages
These assays hold promise for improving early diagnosis, especially in patients who may not have developed a robust antibody response. They are also less likely to yield false positives due to cross-reactivity.
Limitations
T-cell-based assays are still largely experimental and not widely available in clinical practice. More research is needed to validate their use in different stages of Lyme disease and in diverse patient populations.
Once again, there have been reports of cases where initial negative results were followed by subsequent positive findings.
Next-Generation Sequencing (NGS) and Metagenomic Approaches
Next-Generation Sequencing (NGS) has emerged as a promising tool in the diagnosis of infectious diseases, including Lyme disease. Metagenomics, an NGS-based approach, involves the sequencing of all genetic material in a given sample, which allows for the detection of Borrelia DNA without the need for prior knowledge of the specific pathogen.
Advantages
NGS can identify not only Borrelia burgdorferi but also other co-infecting pathogens such as Anaplasma, Babesia, and Bartonella, which are often transmitted by the same ticks. This broad-spectrum detection capability makes it useful in diagnosing patients with atypical or mixed presentations. It also circumvents the need for amplification, as in PCR, making it less prone to technical errors.
Limitations
NGS is not yet widely available for clinical use due to its cost, the complexity of the testing process, and the need for specialized equipment and bioinformatics expertise. Additionally, the sensitivity of NGS can be lower when the bacterial load is low, such as in the early stages of Borreliosis.
Clinical Impact
NGS holds promise for difficult-to-diagnose cases, such as those with neuroborreliosis, where traditional tests may fail. By offering a more comprehensive picture of microbial presence, NGS could lead to earlier and more accurate diagnoses, particularly in patients with overlapping tick-borne infections.
Point-of-Care (POC) Testing
Point-of-care testing is increasingly being explored for the rapid diagnosis of Borreliosis, especially in resource-limited or field settings where access to laboratory facilities may be limited. Several approaches to POC testing are under development, focusing on detecting Borrelia-specific antigens or antibodies within minutes, without the need for sophisticated laboratory equipment.
Lateral Flow Assays (LFAs)
One example of a POC test is the lateral flow assay, which is similar in principle to home pregnancy tests. These devices detect Borrelia antigens or antibodies in blood or other bodily fluids. The advantage of LFAs is their simplicity, allowing for immediate results at the patient's bedside or in field settings. However, their sensitivity and specificity are not yet comparable to laboratory-based tests.
Microfluidics and Biosensors
Another avenue of POC diagnostics involves microfluidic platforms and biosensors that can detect minute concentrations of Borrelia DNA or proteins. These systems are currently in the research phase but could revolutionize Borreliosis diagnosis by enabling highly sensitive detection in a portable format.
Limitations
Although promising, many POC tests are still in the development or validation stages, and their use in clinical practice remains limited. POC tests also face challenges related to sensitivity, particularly in detecting early-stage infection, where antigen or antibody concentrations may be low.
Diagnosis of Neuroborreliosis
Neuroborreliosis, a manifestation of Lyme disease that affects the central and peripheral nervous systems, can be particularly challenging to diagnose. The symptoms are highly variable and can include meningitis, cranial neuropathy (especially facial palsy), radiculopathy, encephalitis, and cognitive disturbances. The diagnostic complexity is compounded by the fact that these neurological symptoms often mimic other neurological disorders, including multiple sclerosis, Guillain-Barré syndrome, or viral encephalitis.
Cerebrospinal Fluid (CSF) Analysis
In cases of suspected neuroborreliosis, analysis of cerebrospinal fluid (CSF) plays a role in diagnosis. The most common findings in CSF include elevated white blood cell count (pleocytosis), increased protein levels, and the potential presence of Borrelia-specific antibodies.
Borrelia-Specific Antibodies in CSF
Detection of intrathecal production of Borrelia-specific IgG antibodies is a key diagnostic marker for neuroborreliosis. The ratio of antibody concentration in the CSF to serum can help differentiate between systemic and CNS involvement. A high CSF-to-serum antibody ratio suggests local production of antibodies within the CNS, which is indicative of neuroborreliosis.
Limitations
CSF analysis, while important, has limitations. Antibodies may take several weeks to appear, so a negative result early in the infection does not rule out neuroborreliosis. Furthermore, patients with late-stage neuroborreliosis may have no detectable antibodies in the CSF.
PCR Testing in CSF
PCR testing of CSF for Borrelia DNA offers a more direct method of detecting infection. This approach is particularly useful in patients with early-stage neuroborreliosis, where antibody tests may be negative.
Advantages
PCR testing provides definitive evidence of Borrelia presence in the nervous system, particularly when antibody production has not yet occurred. PCR is most sensitive when performed on samples taken during the early phase of neurological involvement.
Limitations
As with PCR testing of blood, the bacterial load in the CSF can be low, leading to false-negative results. Thus, a negative PCR result does not definitively rule out neuroborreliosis. Furthermore, obtaining CSF samples requires a lumbar puncture, an invasive procedure with its own risks.
Imaging in Neuroborreliosis Diagnosis
Neuroimaging techniques such as magnetic resonance imaging (MRI) and computed tomography (CT) can aid in the diagnosis of neuroborreliosis, although their utility is more supportive than definitive. MRI can reveal areas of inflammation in the brain and spinal cord, especially in cases of meningoencephalitis or myelitis, which can occur in advanced neuroborreliosis.
MRI Findings
MRI abnormalities are often nonspecific but may include white matter lesions similar to those seen in multiple sclerosis. In cases of meningitis, MRI may show meningeal enhancement. However, such findings are not unique to neuroborreliosis and must be interpreted in conjunction with clinical and laboratory data.
Limitations
Imaging findings alone are insufficient for diagnosis, as many of the abnormalities observed are nonspecific and can be associated with other neurological diseases. Thus, imaging is often used as a complementary tool alongside serological and CSF analysis.
Lyme Disease in Special Populations
Pediatric Lyme Disease: Diagnostic Considerations
Lyme disease in children presents unique diagnostic challenges, as their clinical manifestations and immune responses can differ significantly from those in adults. Early recognition and appropriate diagnostic testing are essential to prevent long-term complications, which can have a more pronounced impact on a child’s development.
Diagnostic Tests in Pediatric Patients
Serological testing remains the cornerstone of diagnosis in children, but some challenges exist. Children may not mount as strong an antibody response as adults in the early stages of infection, leading to false-negative ELISA results. Additionally, the interpretation of Western blot results may be more challenging, as pediatric patients may develop different patterns of antibody responses.
Lyme Disease in Immunocompromised Patients
Patients with compromised immune systems—whether due to HIV, chemotherapy, organ transplantation, or autoimmune conditions—face additional challenges in both the diagnosis and management of Lyme disease. Immunosuppression can alter the body’s response to Borrelia, potentially leading to atypical presentations and diagnostic difficulties.
Altered Immune Response: Immunocompromised patients may not produce detectable levels of Borrelia-specific antibodies, making traditional serological tests like ELISA and Western blot less reliable. These patients may also experience more severe and prolonged infections due to their reduced ability to mount an effective immune response.
References:
- "A Rare Presentation of Lyme Disease in an Immunocompromised Patient", Eric J. Basile, Madeline Smoot, Megan E. Hanna, Zohaib Ijaz, Ellen C. Keeley 2024, DOI: 10.7759/cureus.5860
- Berger, T. G., Schoerner, C., Schell, H., Simon, M., Schuler, G., Röllinghoff, M., et al. (2003). Two unusual cases of diffuse acrodermatitis chronica atrophicans seronegative for Lyme borreliosis. Eur. J. Clin. Microbiol. Infect. Dis. 22, 392–395. doi: 10.1007/s10096-003-0943-1
- The LymeCare Alliance archive also includes records of negative ELISA and Western Blot test results.
Increased Risk of Disseminated Disease
Immunocompromised individuals are at greater risk of developing early disseminated Lyme disease, as their immune systems are less capable of containing the infection locally. As a result, these patients may present with more severe manifestations, including Lyme carditis, arthritis, or neuroborreliosis, often with a faster progression.
Molecular Diagnostics in Immunocompromised Patients
In these populations, molecular diagnostic techniques such as PCR may be more effective than serology. PCR testing of blood, CSF, or synovial fluid may be necessary to detect Borrelia DNA directly. However, the low bacterial load in certain tissues may still result in false-negative PCR results.
Co-infections
Immunocompromised patients are also more susceptible to co-infections with other tick-borne pathogens such as Babesia and Anaplasma.
Lyme Disease in Pregnant Women: Diagnostic and Clinical Challenges
Lyme disease during pregnancy raises concerns due to the potential risks to both the mother and fetus. Although transplacental transmission of Borrelia burgdorferi is rare, untreated maternal Lyme disease has been associated with adverse outcomes, including fetal death, congenital malformations, and preterm birth.
Diagnosis in Pregnant Women
The clinical presentation of Lyme disease in pregnant women is generally similar to that in non-pregnant adults. However, physiological changes during pregnancy, such as altered immune function, can affect the course of infection and the interpretation of diagnostic tests. Pregnant women may have a delayed or reduced antibody response, which can complicate serological testing.
Maternal-Fetal Transmission
While transplacental transmission of Borrelia is rare, cases have been documented. Diagnosis of congenital Lyme disease remains controversial and difficult, as no standardized criteria or diagnostic tests have been established. PCR testing of placental tissue, umbilical cord blood, or amniotic fluid may help identify cases of fetal infection, but the reliability of these tests remains under investigation.
Diagnostic Considerations
Given the potential risks to the fetus, early and accurate diagnosis in pregnant women is crucial. ELISA and Western blot remain the primary diagnostic tools, but clinicians must interpret results cautiously due to the potential for altered immune responses. In some cases, early treatment may be initiated based on clinical suspicion alone, without waiting for confirmatory tests.
Diagnosis and Treatment in Older Adults
Older adults represent another special population where the diagnosis of Lyme disease can be more challenging. The aging immune system, along with the presence of comorbidities and polypharmacy, can obscure the clinical presentation and complicate diagnostic testing.
Atypical Presentation in Older Adults
Older adults may not present with the typical signs of early Lyme disease, such as erythema migrans. Instead, they may exhibit more subtle or non-specific symptoms, including fatigue, cognitive decline, or joint pain. These symptoms often overlap with age-related conditions such as arthritis, dementia, or cardiovascular disease, making the diagnosis of Lyme disease more difficult.
Immune Senescence and Serological Testing
Immune senescence, the gradual deterioration of the immune system associated with aging, can affect the production of antibodies in response to Borrelia infection. As a result, older adults may have weaker antibody responses, leading to false-negative ELISA or Western blot results. Therefore, a negative serological test does not rule out Lyme disease in this population, and PCR testing or clinical diagnosis may be necessary.
Comorbidities and Co-infections
The presence of comorbid conditions such as diabetes, cardiovascular disease, or neurodegenerative disorders can mask the symptoms of Lyme disease or exacerbate them, complicating the diagnostic process. Additionally, older adults may be more susceptible to co-infections with other tick-borne pathogens, further blurring the clinical picture.
Difficulties in Serologic Diagnostics with Borrelia afzelii and Borrelia garinii due to Test Limitations
Diagnosing Lyme borreliosis caused by Borrelia afzelii and Borrelia garinii presents unique challenges, especially in Europe where these strains are more prevalent than Borrelia burgdorferi sensu stricto (which is more common in the U.S.). The primary issue lies in the limitations of current serological tests like ELISA and Western blot, which often fail to accurately detect these species, leading to false negatives and delayed treatment.
Antigenic Variability
One of the main reasons for diagnostic difficulties is the antigenic variability between Borrelia species. Both B. afzelii and B. garinii exhibit differences in surface proteins, such as OspC and flagellin (p41), which are used as targets in serological tests. This variability means that tests developed primarily for Borrelia burgdorferi may not be sensitive enough to detect infections caused by B. afzelii or B. garinii.
As a result, patients with these infections may show negative results despite being infected, especially in the early stages when antibody levels are lower.
Reference: "Comparison of Findings for Patients with Borrelia garinii and Borrelia afzelii Isolated from Cerebrospinal Fluid", 2006, F. Strle, E. Ružić-Sabljić, J. Cimperman, S. Lotrič-Furlan, V. Maraspin
Reference: "Lyme borreliosis diagnosis: state of the art of improvements and innovations", 2023, Mickaël Guérin, Marc Shawky, Ahed Zedan, Stéphane Octave, Bérangère Avalle, Irene Maffucci & Séverine Padiolleau-Lefèvre
"...The species involved in the disease (Bbss in North America and B. garinii, B. afzelii, B. spielmanii, B. bavariensis, in Europe) and the criteria applied for considering the positivity of the diagnosis are different..."
Diagnostic tests developed primarily for Borrelia burgdorferi sensu stricto may not be as effective for detecting Borrelia afzelii or Borrelia garinii. This could lead to false negatives because the tests may not recognize or capture the immune response specific to these European species.
"Serology encounters limitations to detect early localized Lyme disease, as well as early disseminated or late-stage LB."
This statement directly mentions the limitations of serological testing not only for early localized cases but also for disseminated and late-stage Lyme borreliosis. This indicates that even in more advanced stages of the disease, false negatives can occur due to limitations in the testing methods.
"...Bbsl is able to escape the immune system according to a large variety of mechanisms, inducing a reduced immune protection despite the activation of innate and adaptive immunity..."
This highlights that Borrelia species, including B. afzelii and B. garinii, have mechanisms to evade immune detection, which could result in lower antibody production and false negatives, even in later stages of the disease.
Reference: "Borrelia burgdorferi Stimulates Macrophages to Secrete Higher Levels of Cytokines and Chemokines than Borrelia afzelii or Borrelia garinii" 2009, Klemen Strle, Elise E. Drouin, Shiqian Shen, Joseph El Khoury, Gail McHugh, Eva Ruzic-Sabljic, Franc Strle, Allen C. Steere
The study shows that Borrelia burgdorferi induces significantly higher levels of cytokines and chemokines in macrophages than Borrelia afzelii or Borrelia garinii. This stronger inflammatory response may lead to more prominent symptoms and detectable immune reactions in patients infected with B. burgdorferi. Since diagnostic tests, especially serological ones, often rely on the detection of immune responses (such as antibodies or inflammation markers), the weaker immune response to B. afzelii or B. garinii could result in lower detection rates, potentially leading to false negative results, especially in early stages of infection or in cases with less pronounced symptoms.
Immunoblot Limitations
The two-tiered testing system, which involves ELISA followed by Western blot, may also pose challenges in detecting B. afzelii and B. garinii. While Western blot can identify specific antibodies against Borrelia proteins, it is often optimized for B. burgdorferi sensu stricto and may not include antigens most relevant to European species.
This can lead to false-negative results in cases where B. afzelii or B. garinii are the causative agents.
Therefore, patients should ensure that their Borrelia tests are comprehensive, and doctors ordering these tests should verify this as well. Additionally, laboratories and their representatives must provide accurate and complete information about the tests.
Patients should insist on receiving clear and reliable information about what their Borrelia tests include.
Geographical Variation in Borrelia Strains
Serological tests developed for use in one geographical region (such as the U.S.) may not perform as well in other regions where different Borrelia species predominate. B. afzelii is more commonly associated with skin manifestations like acrodermatitis chronica atrophicans (ACA), while B. garinii is linked to neurological symptoms. The variation in clinical presentation adds another layer of complexity to diagnosis
Improving Diagnostic Accuracy
To improve the accuracy of serologic tests for Lyme disease, particularly in regions where B. afzelii and B. garinii are prevalent, researchers recommend using species-specific antigens and developing more sensitive molecular methods like PCR to complement serology. Additionally, adopting new biomarkers and multiplex assays that detect multiple Borrelia species in one test could enhance diagnostic precision