Understanding Lyme Disease Testing: A Patient's Complete Guide to Diagnosis

Table of Contents

• Key Points
• Why This Research Matters
• How This Review Was Conducted
• Understanding the Lyme Bacterium
• Global Varieties of the Lyme Bacterium
• The Range of Lyme Disease Symptoms
• How Doctors Test for Lyme Disease
• Growing the Bacteria: Culture Techniques
• Molecular Detection: PCR Testing
• Antibody Testing: The Body's Immune Response
• The Two-Tier Testing System
• Newer Antibody Tests
• Specialized Antibody Tests
• Cerebrospinal Fluid Testing and Cellular Immunity
• Making Sense of Test Results
• Limitations of Current Testing
• What This Means for Patients
• Frequently Asked Questions
• Source Information

Key Points

• The bull's-eye rash is the key to early Lyme disease diagnosis, while lab tests are vital for joint, nervous, or heart symptoms.
• More than 2.7 million serum samples are tested annually in the US for Borrelia burgdorferi antibodies.
• Lyme disease is transmitted by Ixodes ricinus complex ticks and caused by the spirochete Borrelia burgdorferi.
• The bacterium's genome has a linear chromosome and 21 plasmids, with over 150 lipoprotein genes.
• About 95% of US Lyme disease cases occur in 12 states, with 23,763 cases reported in 2002.

Why This Research Matters

Lyme disease, also known as Lyme borreliosis (LB), is transmitted to humans through the bite of ticks belonging to the Ixodes ricinus complex. The disease was first recognized as a new medical entity in the United States in the late 1970s, although many of its individual symptoms had been documented in Europe decades earlier. The bacterium responsible for the illness, Borrelia burgdorferi, was first isolated in 1982 from the tick vector Ixodes dammini (now called Ixodes scapularis), and subsequently from skin biopsies, cerebrospinal fluid (CSF — the fluid surrounding the brain and spinal cord), and blood samples of patients with Lyme disease.

The Centers for Disease Control and Prevention (CDC) began tracking Lyme disease cases in 1982, and in 1990 it became a nationally notifiable disease — meaning health professionals are required to report cases to public health authorities. Since 1982, more than 200,000 cases have been reported in the United States, with about 17,000 cases reported each year between 1998 and 2001. In 2002, the number of cases increased to 23,763, with a national incidence of 8.2 cases per 100,000 people.

Approximately 95% of cases occur in just 12 states located in the northeastern, mid-Atlantic, and north central regions of the country. These states are: Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Wisconsin. Lyme disease is also widely distributed in European countries, and it occurs in far eastern Russia and in some Asian countries.

One especially striking statistic from this review: more than 2.7 million blood serum samples are tested each year in the United States alone for B. burgdorferi-specific antibodies. This enormous testing demand has driven the development of many new diagnostic tools, including tests that directly detect the bacterium and tests that identify antibodies using whole-cell preparations, recombinant antigens, or synthetic peptide antigens in enzyme immunoassays (EIAs).

How This Review Was Conducted

This article is a comprehensive medical review written by four experts from the New York Medical College and Westchester Medical Center in Valhalla, New York. Rather than reporting the results of a single new study, the authors analyzed and synthesized decades of published research on laboratory testing for Lyme disease. They examined studies on the biology of B. burgdorferi, the clinical features of the illness, direct detection methods (culture and molecular techniques), and immunologic approaches such as antibody testing.

Their goal was to provide a complete assessment of currently available laboratory tests for Lyme borreliosis, explain how to interpret test results, and discuss future directions for improving established tests and developing new approaches. The review draws on more than 375 published references, making it one of the most thorough examinations of Lyme disease diagnosis available at the time of its publication.

Understanding the Lyme Bacterium

B. burgdorferi is a spiral-shaped (helically shaped) bacterium with multiple internal flagella — the tail-like structures that allow it to move. The cells have 3 to 10 loose coils, are 10 to 30 micrometers in length, and 0.2 to 0.5 micrometers in width. This spirochete (a term for spiral-shaped bacteria) possesses several features that are distinctive among bacteria.

Live B. burgdorferi organisms are motile and can swim. In the laboratory, they can be visualized using dark-field or phase-contrast microscopy (specialized microscopes that illuminate unstained organisms). They can also be seen under a standard light microscope after staining with silver stains or using fluorescent microscopy methods. The bacterium's structure includes an outer slime layer, a three-layered outer membrane surrounding a space that contains 7 to 11 flagella, and an inner compartment called the protoplasmic cylinder.

B. burgdorferi was the first spirochete whose complete genome (its entire genetic blueprint) was sequenced. The genome of the standard laboratory strain, B. burgdorferi sensu stricto B31, is 1,521,419 base pairs (the chemical building blocks of DNA). It consists of a linear chromosome of 910,725 base pairs, with a guanine-cytosine (G+C) content of 28.6%, plus 21 plasmids — small, separate DNA molecules — of which 9 are circular and 12 are linear. Together, these plasmids have a combined size of 610,694 base pairs.

Comparative analysis of the genome of a recently sequenced strain of a related species, Borrelia garinii strain PBi, revealed that most of the chromosome is conserved (meaning it has stayed largely the same through evolution), with 92.7% identity at both the DNA and amino acid levels. The chromosome and two linear plasmids (lp54 and cp26) carry approximately 860 genes that appear to form the basic genetic inventory of Lyme Borrelia species.

Genome analysis revealed several unusual genetic features that are uncommon among other bacteria:

• A linear chromosome and multiple linear and circular plasmids in a single bacterium
• A unique organization of the ribosomal RNA gene cluster (the genes that help build the cell's protein-making machinery)
• Over 150 lipoprotein-encoding genes (proteins attached to fat molecules), accounting for 4.9% of the chromosomal genes and 14.5% of the plasmid genes — a proportion significantly higher than any other bacterial genome sequenced to date
• A substantial portion of plasmid DNA that appears to be in a state of evolutionary decay
• Evidence for numerous, potentially recent DNA rearrangements among the plasmid genes
• A lack of genes that encode the enzymes needed to synthesize amino acids, fatty acids, enzyme cofactors, and nucleotides
• A lack of genes for the tricarboxylic acid cycle (a key energy-production pathway) and for compounds involved in electron transport

Together, these genetic findings indicate that B. burgdorferi has a parasitic lifestyle, relying on its host for many essential nutrients and building blocks.

One of the most fascinating aspects of this bacterium is its ability to change which surface proteins it expresses depending on its environment. For example, the spirochete produces the outer surface protein A (OspA) but not OspC when residing in the midguts of unfed ticks. However, when the tick takes a blood meal, some spirochetes stop expressing OspA and instead express OspC — a key protein involved in establishing infection in a mammal. Certain other genes are expressed only in a mammalian host or have significantly increased expression in that environment; these include the genes for VlsE, DbpA, BBK32, Erp, and Mlp proteins.

In a whole-genome microarray experiment, researchers grew B. burgdorferi at 23°C and 35°C to simulate the temperatures found in tick vectors and mammalian hosts, respectively. They found that a total of 215 open reading frames (segments of genes) were differentially expressed between the two temperatures. Strikingly, 136 (63%) of the differentially expressed genes were carried on plasmids, highlighting the potential importance of plasmid genes in helping the bacterium adapt to diverse environmental conditions.

Global Varieties of the Lyme Bacterium

Eleven species within the B. burgdorferi sensu lato complex (the broader group of closely related Lyme disease bacteria) have been described worldwide. The distribution varies by continent:

• North America: Three species have been identified — 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, and Korea): Seven species — B. garinii, B. afzelii, B. valaisiana, Borrelia japonica, Borrelia tanukii, Borrelia turdi, and Borrelia sinica

At least three species are definitely capable of causing human disease in Europe: B. burgdorferi sensu stricto, B. garin

Frequently Asked Questions