Table of Contents
- Key Points
- Why Lyme Disease Research Matters
- The Bacterium Behind Lyme Disease
- Inside the Genome: Unusual Features
- The Many Species of Lyme Bacteria
- How the Bacterium Changes Its Coat
- The Full Spectrum of Lyme Disease
- Laboratory Diagnosis: The Big Picture
- Growing the Bacteria: Culture Techniques
- Which Patient Samples Can Be Cultured
- Limitations of This Research
- What This Means for Patients
- Frequently Asked Questions
- Source Information
Key Points
- Lyme disease is caused by Borrelia burgdorferi, a spirochete transmitted by Ixodes ticks, with about 95% of U.S. cases in 12 states.
- Culture of the bacterium is the strongest proof of active infection but takes up to 12 weeks and requires specialized media and skilled microscopists.
- Erythema migrans, the expanding rash of at least 5 cm, appears in at least 80% of patients with objective evidence and can be diagnosed clinically.
- Up to 10% of patients with Lyme arthritis may have joint inflammation lasting months or years despite antibiotics, more common with certain HLA DRB variants.
- Different Borrelia species cause disease in North America, Europe, and Asia, leading to regional differences in late manifestations like arthritis or ACA.
Why Lyme Disease Research Matters
Lyme borreliosis (LB) — the medical name for Lyme disease — is transmitted by ticks of the Ixodes ricinus complex. These are the same hard-bodied ticks, sometimes called deer ticks, that feed on people in wooded areas. The illness was first described as a distinct condition in the United States in the late 1970s.
Since 1982, when public health tracking began, more than 200,000 cases have been reported to the U.S. Centers for Disease Control and Prevention (CDC). Between 1998 and 2001, roughly 17,000 cases were reported each year. In 2002, that number rose to 23,763 cases, giving a national incidence of 8.2 cases per 100,000 people.
Cases are highly concentrated geographically. About 95% of all cases occur in just 12 states: Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin. That cluster sits in the northeastern, mid-Atlantic, and north central regions of the country.
Lyme disease is not only a North American problem. It is widely distributed across European countries, and it also occurs in far eastern Russia and parts of Asia. Because the disease is so common and so geographically widespread, the demand for reliable laboratory testing is enormous. In the United States alone, an estimated more than 2.7 million serum samples are tested every year for antibodies against B. burgdorferi.
To meet that demand, laboratories have introduced many different tests. Some detect the bacterium directly. Others look for the immune system's antibodies using whole-cell lysates (broken-up bacterial material), recombinant antigens (lab-made proteins), or peptide antigens (short protein fragments). These tests are enzyme immunoassays (EIA, a test that uses a color change to detect a reaction). This review assesses how those tests were developed and how well they perform.
The Bacterium Behind Lyme Disease
The causative agent, Borrelia burgdorferi, is a spirochete — a helically shaped (corkscrew-like) bacterium with multiple endoflagella (internal whip-like tails that let it swim). The cells are configured with 3 to 10 loose coils and measure 10 to 30 micrometers (µm) in length and 0.2 to 0.5 µm in width.
The organism was first recovered in 1982 from the vector tick Ixodes dammini, now renamed Ixodes scapularis. It was subsequently grown from skin biopsy specimens, cerebrospinal fluid (CSF, the fluid surrounding the brain and spinal cord), and blood samples. These samples came from patients in both the United States and Europe.
Cultured B. burgdorferi organisms are motile and swim when placed on freshly prepared slides. Live organisms can be seen using dark-field or phase-contrast microscopy — techniques that make thin, transparent objects visible without staining. They can also be recognized under a standard light microscope after staining with silver stains, or with fluorescent microscopic methods.
The structure of the bacterium has several layers. Its ultrastructure (fine internal anatomy) includes:
- An outer slime surface layer (called the S-layer)
- A trilaminar outer membrane surrounding the periplasmic space (the gap between the inner and outer membranes)
- 7 to 11 periplasmic flagella inside that space
- An innermost compartment called the protoplasmic cylinder
Inside the Genome: Unusual Features
B. burgdorferi was the first spirochete to have its complete genome (its entire set of genetic instructions) sequenced. The genome of the reference strain, B. burgdorferi sensu stricto B31, totals 1,521,419 base pairs (bp) — the individual "letters" of genetic code.
That total splits into a linear chromosome of 910,725 bp, with a G+C content (a measure of the chemical makeup of DNA) of 28.6%, plus 21 plasmids — 9 circular and 12 linear — with a combined size of 610,694 bp. Plasmids are small, separate loops or strands of DNA that sit outside the main chromosome.
Comparing this genome to that of a related European species, Borrelia garinii strain PBi, researchers found most of the chromosome is conserved: 92.7% identity in both DNA and amino acid sequence. The chromosome plus two linear plasmids (lp54 and cp26), which carry approximately 860 genes, appear to form the basic genetic inventory of all Lyme Borrelia species. Not every strain of B. burgdorferi has the full set of plasmids, so total genome size varies between isolates.
Genome analysis revealed several features that are uncommon among bacteria:
- A linear chromosome and multiple linear and circular plasmids
- A unique organization of ribosomal RNA genes: a single 16S rRNA gene (rrs) with tandemly repeated 23S (rrl) and 5S (rrf) rRNA genes
- More than 150 lipoprotein-encoding genes, accounting for 4.9% of chromosomal genes and 14.5% of plasmid genes — significantly higher than any other bacterial genome sequenced at that time
- A substantial fraction of plasmid DNA that appears to be in a state of evolutionary decay
- Evidence of numerous, potentially recent DNA rearrangements among plasmid genes
- A lack of recognizable genes for making amino acids, fatty acids, enzyme cofactors, and nucleotides
The bacterium also lacks genes for tricarboxylic acid cycle enzymes (part of the cell's energy-producing machinery) and for compounds involved in electron transport. Taken together, these missing genes indicate that B. burgdorferi is parasitic by nature — it depends on its host for many basic building blocks rather than manufacturing them itself.
The Many Species of Lyme Bacteria
Eleven Borrelia species within the B. burgdorferi sensu lato complex (the broader group of related Lyme bacteria) have been described worldwide. They are not distributed evenly.
- North America: three species — B. burgdorferi sensu stricto, Borrelia andersonii, and Borrelia bissettii
- Europe: five species — B. burgdorferi sensu stricto, B. garinii, Borrelia afzelii, Borrelia valaisiana, and Borrelia lusitaniae
- Asia (China, Japan, Korea): seven species — B. garinii, B. afzelii, B. valaisiana, Borrelia japonica, Borrelia tanukii, Borrelia turdi, and Borrelia sinica
Not all of these species make people sick. In Europe, at least three are known to be pathogenic (disease-causing) in humans: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. In the United States, B. burgdorferi sensu stricto is the only species known to cause human infection.
Within B. burgdorferi sensu stricto, researchers have identified several subtypes. Studies have linked specific subtypes to hematogenous dissemination (spread of the bacteria through the bloodstream) and tissue invasion in patients, as well as in experimentally infected animals.
In Asia, only B. garinii and B. afzelii have been definitively confirmed as human pathogens. Studies in patients and laboratory animals suggest that B. bissettii, B. valaisiana, and B. lusitaniae may also be capable of causing disease, but their ability to infect humans is not well established.
Genetic comparisons show these species are closely related, with highly conserved chromosome gene orders and similar plasmid profiles. More recent studies, however, have documented meaningful genetic heterogeneity (variation) among isolates both in the United States and in Europe. A large number of B. burgdorferi sensu lato gene sequences are now available in GenBank, the public database maintained by the National Center for Biotechnology Information, including fla, vlsE, bmpA, and dbpA, plus genes for 16S rRNA and outer surface proteins A and C.
How the Bacterium Changes Its Coat
B. burgdorferi sensu lato expresses different surface proteins depending on where it is living — a survival strategy for adapting to different environments inside ticks and mammals. This matters enormously for diagnosis.
When the spirochete resides in the midguts of unfed ticks, it displays OspA but not OspC. During a tick's blood meal, some spirochetes stop making OspA and begin making OspC instead. Other genes are expressed only in a mammalian host, or are significantly ramped up there. These include VlsE, DbpA, BBK32, Erp, and Mlp proteins.
Researchers have used whole-genome microarrays (tools that measure the activity of thousands of genes at once) to study gene expression under conditions mimicking unfed ticks, fed ticks, and mammalian hosts. In one key experiment, B. burgdorferi B31 was grown at 23°C and 35°C, simulating the temperatures found in tick vectors and mammalian hosts, respectively. A total of 215 open reading frames (gene sequences) were differentially expressed at the two temperatures. Strikingly, 136 of these — 63% — were carried on plasmids. That finding highlights how important plasmid-carried genes are for adjusting to different environments.
Genetic diversity and differential gene expression have direct consequences for laboratory testing. The choice of PCR primers (short DNA sequences used to detect bacterial genetic material) can target different segments of the genome. The selection of particular antigens for antibody tests can also change how sensitive and specific a diagnostic assay is.
The Full Spectrum of Lyme Disease
Infection with B. burgdorferi sensu lato can produce skin, neurological, cardiac, and musculoskeletal disorders. The basic clinical picture is similar worldwide, but there are well-documented differences between Europe and North America. Those differences are attributed to the different bacterial species causing disease on each continent, and even within Europe, patterns vary by region depending on which species dominates.
Early infection usually begins with localized erythema migrans (EM) — the expanding red rash often called the "bull's-eye" rash. Within days or weeks, it may be followed by clinical evidence of disseminated infection affecting the skin, nervous system, heart, or joints. Months later, late infection can appear.
EM is the characteristic sign of early infection and the clinical hallmark of Lyme disease. In recent series, it is recognized in at least 80% of patients who have objective clinical evidence of infection meeting the CDC's surveillance definition. The rash starts at the site of the tick bite as a red macule or papule (a flat or slightly raised spot), rapidly enlarges, and sometimes develops central clearing.
The clinical diagnosis of early Lyme disease with EM relies on recognizing the characteristic appearance of a skin lesion at least 5 cm in diameter. At this stage, patients may have no symptoms at all. More commonly in the United States, patients may experience flu-like symptoms such as headache, myalgia (muscle aches), arthralgias (joint pain), or fever.
Some investigators treat the combination of constitutional symptoms plus EM as evidence of dissemination, but the authors note this is not evidence based. They prefer to call this presentation "symptomatic EM" instead.
Hematogenous dissemination (spread through the bloodstream) can reach the nervous system, joints, heart, or other skin areas. Occasionally, hematogenous dissemination reaches other organs, and it can produce a wide range of early Lyme disease manifestations. Patients with objective evidence of dissemination usually experience one or more of the following syndromes:
- Multiple EM lesions
- Atrioventricular conduction defects (disrupted electrical signaling in the heart)
- Myopericarditis (inflammation of the heart muscle and surrounding sac)
- Arthritis
- Facial palsy (weakness of the facial nerve)
- Meningitis (inflammation of the membranes around the brain and spinal cord)
- Meningoradiculoneuritis, also known as Bannwarth's syndrome (nerve root inflammation)
Late Lyme disease may develop in some untreated patients months to a few years after tick-transmitted infection. Its major manifestations are arthritis, late neuroborreliosis (peripheral neuropathy or encephalomyelitis — nerve damage or brain and spinal cord inflammation), and acrodermatitis chronica atrophicans (ACA, a slowly progressive skin condition).
Lyme arthritis begins as intermittent attacks of mono- or pauciarticular arthritis (affecting one or a few joints), especially large joints. In up to 10% of patients, arthritis may persist for months or a few years despite treatment with antimicrobials (antibiotics and related drugs). Treatment-resistant arthritis is seen more frequently in patients with certain HLA DRB alleles (inherited immune-system gene variants). Researchers have suggested that autoimmunity — the immune system attacking the body's own tissues — plays a role in this form.
Where a patient lives shapes what late disease looks like. Lyme arthritis is the most common late manifestation in North America, while ACA appears to be the most common late manifestation in Europe. Again, these differences are likely due to the different bacterial species causing infection on each continent.
Laboratory Diagnosis: The Big Picture
A variety of laboratory techniques have been developed for directly detecting B. burgdorferi sensu lato. These assays provide evidence of intact spirochetes, or of spirochete components such as DNA or protein, in tick vectors, reservoir hosts (animals that carry the bacteria in nature), or patients.
Four different approaches have been used in the clinical laboratory:
- Microscope-based assays
- Detection of B. burgdorferi-specific proteins
- Detection of B. burgdorferi-specific nucleic acids (DNA)
- Culture (growing the organism)
Of these, culture of B. burgdorferi sensu lato undoubtedly offers the best confirmation of active infection, and it has been increasingly used as a diagnostic tool by researchers on both sides of the Atlantic. Growing the organism also lets scientists study the structural, molecular, antigenic, and disease-causing properties of the different species.
Direct microscopic detection of the bacterium has limited clinical usefulness for confirming Lyme disease, because organisms are so sparse in clinical samples. Antigen detection assays other than PCR suffer from the same problem. Antigen capture tests have been used to detect bacterial antigens in the CSF of patients with neuroborreliosis. These tests have also been used in urine samples from patients with suspected Lyme disease, but their reliability is poor or, at best, questionable.
Growing the Bacteria: Culture Techniques
The liquid media (nutrient solutions) used to grow B. burgdorferi sensu lato today were derived from the original Kelly medium through a series of modifications over time. Current versions include:
- Barbour-Stoenner-Kelly II medium (BSK II) — uses CMRL-1066 without glutamine, plus Yeastolate, neopeptone as the peptone preparation, and HEPES as a buffer
- BSK-H — omits gelatin and uses different proportions of certain ingredients
- Kelly medium Preac-Mursic (MKP) — removes Yeastolate and uses different proportions of certain ingredients; also called the Pettenkofer modification
- Stoerner modification — adds Yeastolate and CMRL-1066, without glutamine and without sodium bicarbonate
These modern media support growth better than older versions. They allow recovery from low inocula (small starting numbers of bacteria), produce shorter generation times, and reach maximal spirochete concentrations of about 108 to 109 organisms per milliliter.
Key ingredients of BSK II include CMRL-1066, a standard medium used for growing mammalian cells. Other ingredients include bovine serum albumin fraction V, a rich protein source that also stabilizes pH, and N-acetylglucosamine, a building block for the bacterial cell wall. BSK II also includes rabbit serum, citrate, pyruvate, and many other components. The growth-promoting ability of these media depends on careful selection of key ingredients, which can vary widely in composition.
Incubation conditions matter too. Temperatures of 39°C (about 102°F) or higher may reduce or prevent growth. Cultures are incubated for up to 12 weeks — far longer than is needed for most other human bacterial pathogens. That long wait reflects the spirochete's prolonged generation time of 7 to 20 hours or longer during log-phase growth.
Laboratories detect growth by periodically examining a sample of the culture fluid for spirochetes. Laboratories use either dark-field microscopy or fluorescence microscopy after staining with acridine orange dye or a specific fluorescent-labeled antibody. Any spirochete-like structures seen must be confirmed as B. burgdorferi sensu lato, either by showing reactivity with specific monoclonal antibodies (identical, lab-made immune proteins) or by detecting specific DNA sequences using PCR.
Experience matters here. A lack of familiarity with microscopic detection of B. burgdorferi sensu lato can lead to false-positive readings, because other structures such as cellular debris may look thread-like and be mistaken for the bacteria.
The organism can also be grown on solid media, with agarose added to solidify the liquid medium, and incubated under microaerophilic (low-oxygen) or anaerobic (oxygen-free) conditions. One advantage of solid media is that individual colonies can be identified, allowing researchers to isolate particular clonal strains.
Laboratory-propagated strains of B. burgdorferi sensu lato can also be cocultivated (grown together) with tick cell lines and with certain mammalian cell lines. These cocultivation techniques may prove useful for primary isolation of the bacterium directly from clinical specimens.
Which Patient Samples Can Be Cultured
B. burgdorferi sensu lato can be recovered from a variety of tissues and body fluids from patients with Lyme disease. These include:
- Biopsy and lavage (washing) specimens from EM skin lesions
- Biopsy specimens from ACA skin lesions
- Biopsy specimens from borrelial lymphocytoma skin lesions (a rare, benign skin nodule associated with Lyme disease)
- Cerebrospinal fluid
- Blood
This breadth of possible sample types is important, because it means the recovery of the organism depends heavily on which tissue is sampled and at what stage of illness. The original scientific review continues from this point into detailed discussions of molecular detection methods. These methods include PCR and real-time quantitative PCR. The review also covers immunologic diagnosis. It also covers antibody detection methods such as immunofluorescence assays. The review also discusses enzyme immunoassays, Western immunoblotting, two-tier testing, and newer tests using recombinant and peptide antigens. It further covers borreliacidal antibody assays, detection of antibodies bound to circulating immune complexes, antibody testing in cerebrospinal fluid, and cellular immune response tests.
Limitations of This Research
This review describes the state of diagnostic testing as of 2005, so the technologies discussed — and the performance figures quoted — reflect what was available at that time. Newer methods have entered the laboratory since then.
Several limitations run through the whole field. Direct microscopic detection is limited by how few organisms are present in patient samples. Antigen detection tests outside of PCR have poor or questionable reliability. Culture, though the strongest confirmation of active infection, requires up to 12 weeks, needs specialized media and skilled microscopists, and can produce false-positive readings when staff lack experience. Even the bacterial genome varies between strains. Gene expression shifts depending on whether the organism is in a tick or a mammal — factors that can change how well a given diagnostic test performs.
The review also notes areas where knowledge remains unsettled. The ability of B. bissettii, B. valaisiana, and B. lusitaniae to cause human disease is not well established. And the idea that constitutional symptoms alongside EM prove the infection has disseminated is described by the authors as not evidence based.
What This Means for Patients
You may live in the northeastern, mid-Atlantic, or north central United States. You may have traveled to these regions. You may also live in or have traveled to Europe or parts of Asia where Lyme disease occurs. Understanding the diagnostic picture can help. Understanding the diagnostic picture can help you have a better conversation with your doctor.
- Know the rash. Erythema migrans appears in at least 80% of patients with objective evidence of Lyme disease. A skin lesion of at least 5 cm that enlarges over days is the classic sign and can be diagnosed clinically, without a laboratory test.
- Expect a clinical diagnosis first. When the characteristic rash is present, doctors can often diagnose early Lyme disease by its appearance alone.
- Understand that culture takes time. Growing the bacterium is the strongest proof of active infection, but cultures are held for up to 12 weeks because the organism divides slowly — roughly every 7 to 20 hours.
- Ask which sample is being tested. The bacterium is found in different body sites at different stages — skin biopsies of EM, ACA, or lymphocytoma lesions, cerebrospinal fluid, and blood. The right sample depends on your symptoms.
- Ask about the test's limitations. Microscopy and non-PCR antigen tests can miss the organism or produce unreliable results, and reading cultures requires experience to avoid false positives.
- Report persistent joint symptoms. Up to 10% of patients with Lyme arthritis may have joint inflammation that lasts months or years despite antibiotic treatment. This is more common in people with certain inherited HLA DRB gene variants.
The bottom line: Lyme disease is common, geographically concentrated, and caused by a genetically complex organism that changes its surface proteins as it moves between ticks and humans. That complexity is exactly why no single laboratory test is perfect, and why diagnosis still depends on combining your symptoms, your exposure history, and carefully chosen laboratory results.
Frequently Asked Questions
What is the most reliable way to confirm an active Lyme infection?
Growing the bacterium in culture is the strongest proof of active infection. However, it is slow and technically demanding, requiring up to 12 weeks because the organism divides roughly every 7 to 20 hours. It also needs specialized media and skilled microscopists to avoid false-positive readings.
How long does a Lyme culture take, and why?
Cultures are held for up to 12 weeks. This long wait is because the bacterium has a prolonged generation time of 7 to 20 hours or longer during log-phase growth. Laboratories check periodically for growth using dark-field or fluorescence microscopy, and any spirochete-like structures must be confirmed with specific antibodies or DNA testing.
Which body samples can be tested for Lyme bacteria?
The bacterium can be recovered from several tissues and fluids: skin biopsies from erythema migrans, acrodermatitis chronica atrophicans, or borrelial lymphocytoma lesions; cerebrospinal fluid; and blood. The right sample depends on your symptoms and stage of illness, because the organism is found in different body sites at different times.
Can I be diagnosed with Lyme disease without a lab test?
Yes. When the characteristic erythema migrans rash is present, doctors can often diagnose early Lyme disease by its appearance alone. The rash appears in at least 80% of patients with objective evidence of infection, and a skin lesion of at least 5 cm that enlarges over days is the classic sign.
What does it mean if my Lyme arthritis doesn't improve with antibiotics?
Up to 10% of patients with Lyme arthritis may have joint inflammation that lasts months or years despite antibiotic treatment. This treatment-resistant form is seen more frequently in people with certain inherited HLA DRB gene variants, and researchers have suggested that autoimmunity plays a role in this type of arthritis.
Why do Lyme disease symptoms differ between North America and Europe?
Different Borrelia species cause infection on each continent. In North America, B. burgdorferi sensu stricto is the only species known to cause human infection, and Lyme arthritis is the most common late manifestation. In Europe, three species are pathogenic, and acrodermatitis chronica atrophicans is the most common late manifestation.
Are antigen tests or microscopy reliable for diagnosing Lyme disease?
Direct microscopic detection has limited clinical usefulness because organisms are sparse in clinical samples. Antigen detection assays other than PCR suffer the same problem. Antigen capture tests used on cerebrospinal fluid or urine have poor or questionable reliability. Culture remains the strongest confirmation of active infection, though it is slow and technically demanding.
I have a negative Lyme serology but ongoing joint and heart symptoms — when should I seek a second opinion on my Lyme disease diagnosis?
A negative antibody test does not rule out Lyme disease, because test performance shifts with the bacterial species, the antigens chosen, and the stage of illness. Culture of the bacterium is the strongest confirmation of active infection, but it takes up to 12 weeks and needs specialized media and skilled microscopists. If your symptoms persist despite a negative result, or if joint inflammation lasts months after antibiotics, an independent review can help clarify the picture. An independent review of your exposure history, symptoms, and laboratory results can help clarify the picture. Diagnostic Detectives Network provides independent expert second opinions.
Source Information
Original article title: Diagnosis of Lyme Borreliosis
Publication details: Clinical Microbiology Reviews, July 2005, Volume 18, Number 3, pages 484–509. Copyright © 2005, American Society for Microbiology. DOI: 10.1128/CMR.18.3.484–509.2005.
This patient-friendly article is based on peer-reviewed research.
