History and Metabolic Context

Definition and Symptoms of Disease

Lyme disease, also known as Lyme borreliosis, is a bacterial infection one can get from the bite of an infected tick known as Ixodes scapularis. It is now the most commonly reported arthropod-borne illness in the US and Europe and is also found in Asia. The causative agent of Lyme disease is the spirochete bacterium known as Borrelia burgdorferi. Ticks are able to attach to any part of the human body where it may bite and transmit the Lyme disease bacterium to the human host after about 36-48 hours. Most humans are infected by ticks in their nymphal stage, where they are very small (less than 2 millimeters), making them extremely difficult to see or detect. Adult ticks can also transmit Lyme disease bacteria, but because they are larger than nymphs they are more likely to be discovered and removed before they can transmit the disease (CDC Lyme disease transmission).

Within about 3-32 days of infection, a slowly expanding skin lesion, called erythema migrans (EM), forms at the site of the tick bite in about 70-80% of cases of Lyme disease (Smith et al. 2002). This skin lesion is often accompanied with flu-like symptoms, such as fatigue, headache, fever, and by signs that suggest dissemination of the spirochete (Steere et al. 2004). After having been infected for a few weeks, if the bacteria have not been killed off by the body’s own immune system or by prescribed antibiotics, it will often spread throughout the body by means of blood and other fluid transport. During this time, the spirochete has been recovered from blood and cerebrospinal fluid and has also been seen in the retina, muscle, bone, spleen, liver, meninges, and the brain (Steere et al. 2004).

In order to disseminate, the bacteria often bind to extracellular proteins on host cells or tissue matrices. Despite the immune system being very active during dissemination, B. burgdorferi may survive by changing antigenic expression of surface proteins and inhibiting certain host immune responses. As the bacteria spread throughout the body, patients may experience persistent infection, which include symptoms of arthritis, heart conditions, and neurological problems.

History of Discovery

Although Lyme disease is a relatively new diagnosis, reports of the dermatological manifestations of the disease, such as EM, date back to 1883 when Buchwald first observed the skin manifestation considered to be the third stage of disease. Then around 1913, it was observed that an expanding rash, now known as EM, occurred in a patient after they were bitten by a sheep tick. It was not until 1930 that neurologic symptoms were associated with EM and in 1948 it was speculated that the disease was of bacterial origin and doctors began treating EM patients with penicillin, which was shown to be about 70% successful at curing patients with late manifestations of the disease (Steere et al. 1987 and Bhate et al. 2011).

Lyme disease was defined in 1975 because of geographic clustering of children in Lyme, Connecticut who were thought to have juvenile rheumatoid arthritis (Steere et al. 1987). These children often experienced recurring swelling and pain in a few large joints, especially the knee. It then became apparent that a previous annular skin lesion, now known as erythema migrans, was usually the first manifestation of the illness and that the following arthritis symptoms were a manifestation of the disease (Steere et al. 1987). However, the cause of the disease was still unknown.

In 1982, Dr. Burgdorfer and his team of scientists were the first to discover the previously unknown spirochetal bacterium, called Borrelia burgdorferi, in a tick species, known as Ixodes scapularis (Burdgorfer 1982). This spirochete, which is a spiral shaped bacteria, was isolated from the midgut of a tick vector and was later found in patients with early Lyme disease. Patients’ immune responses were also linked with the bacterium, proving it was the cause of disease (Steere et al. 2004). The genomic sequencing of the first strain of this spirochete was completed in 1997 (Fraser et al. 1997). Since its discovery, there has been a lot of research conducted investigating how Borrelia burgdorferi infects humans and how it causes disease symptoms.

Epidemiology in America Today

Lyme is considered to be an emerging disease in the US. During the European colonization of North America, many woodland areas in New England were cleared for farming and deer were hunted almost to extinction (Steere et al. 2004). However in the 20th century as farmland reverted back to woodland, deer, mice, and the deer tick were able to thrive, especially near rivers and along the coast (Steere et al. 2004). Eventually these areas became very populated by humans and deer. During the past 40 years, the infection has spread in the northeastern US and now affects suburban locations near Boston, New York, Philadelphia, and Baltimore (Steere et al. 2004).

The reported incidence of Lyme disease has increased dramatically over the last thirteen years. In 2000, the Centers for Disease Control and Prevention (CDC) reported 17,730 cases of Lyme disease in the United States. This was a slight decrease from the previous year, but a twofold increase over the number of cases reported in 1990. In 2006, there were 19,931 cases reported to the CDC, representing a 101% increase in the 15-year period between 1992 and 2006 (Bhate et al. 2011). More than 90% of the cases occurred in the eastern US and New Jersey had reported 24 cases per 100,000 people. There have also been sporadic cases observed in California and Oregon. Evidence suggests a rise in infection rates. However, this may be attributed to heightened awareness (Bhate et al. 2011).

Incidence of Lyme disease per 100,000 people in each state of the USA in 2009
Incidence of Lyme disease per 100,000 people in each state of the USA in 2009. Alao et al. 2012

Disease Identification/ Diagnosis

            Unfortunately, physicians struggle to diagnose Lyme disease and increased public awareness has led to an over diagnosis and in many cases unnecessary treatment. Most cases of flu-like summertime illnesses are not caused by Lyme disease (Bhate et al. 2011). Lyme disease is reportable in the United States by physician diagnosed erythema migrans greater than 5 cm in diameter or one or more objective late manifestations of Lyme disease with laboratory evidence of infections with B. burgdorferi in an individual with possible exposure to infected ticks (Marques 2010).

Most laboratory testing methods for diagnosis of Lyme disease are indirect and mostly based on serologic assays because of the difficulty in demonstrating B. burgdorferi by direct techniques such are culture and PCR analysis (Marques 2010). Culture of B. burgdorferi from clinical samples has been very useful in better understanding the disease, but has its drawbacks when used for diagnosis. Culture of B. burgdorferi requires special enriched bacterial media and about a 12 week period of observation due to the slow multiplication of B. burgdorferi. The bacteria can be identified in about 50% of cultures of biopsy specimens taken from untreated erythma migrans lesions and in only about 40% in blood samples from patients who have early disease and have not received antibiotics (Marques 2010).

PCR has been used to amplify genomic DNA of B. burgdorferi in skin, blood, and synovial fluid. It seems to be most useful in patients with Lyme arthritis and bacteria have been identified in synovial fluid in about 85% of patients (Marques 2010). PCR sensitivity varies from 25% to 90% in skin biopsies from erythema migrans. PCR of urine samples has been used with variable results as well.

Serologic testing is the most commonly used laboratory test in patients. The sensitivity of the test increases with the duration of the infection because it relies on antibody production. This presents a problem for patients who present early erythma migrans because only about 50% test positive for Lyme disease. Another complication with serologic testing is that is does not distinguish between active and inactive forms of the disease. Therefore, a two-tier serologic testing is often used, in which the first tier is usually a sensitive ELISA using purified or recombinant antigens of B. burgdorferi. If this test is positive or equivocal the second tiered test is to perform a Western Blot using IgM and IgG antibodies. An IgM Western Blot is considered positive if 2 of 3 bands are present (23, 39, and 41 kDa). An IgG blot is considered positive if 5 of 10 bands are present (Alao 2012). These tests are not the best and are not always definitive, which is why there is a need for the development of new or better testing options.

There have been recent attempts to improve diagnosis methods and efficiency. In 2011, three different serologic testing strategies were compared (Branda et al. 2011). Before the comparison was made it was known that a single enzyme immunoassay (EIA) using the C6 peptide of the VlsE expressed lipoprotein provided similar or better sensitivity, but less specificity compared to 2-tiered testing with whole-cell sonicate EIA followed by IgM/IgG Western blots. The authors used an alternative 2-tiered strategy, in which the first step remained a whole cell sonicate EIA, but the Western blot was replaced by a C6 EIA. They found that the 2 EIAs provided sensitivity comparable to that of the C6 EIA but maintained the specificity of standard 2-tiered testing (Branda et al. 2011). It is clear that finding a good diagnosis technique for Lyme disease is quite difficult.

A more recent and very different approach to Lyme detection was made by Lerner et al. in 2013. They examined the potential of antibody-funcionalized single walled carbon nanotube field-affect transistors to use as a fast and accurate sensor for Lyme disease antigen. They attached Lyme flagellar antibodies to nanotubes and measured the binding of Lyme proteins. They were able to detect antigen at concentrations as low as 1 ng/ml. The speed and sensitivity of the biosensor make it an ideal candidate for development as a medical diagnostic test (Lerner et al. 2013).

Normal Function of Immune System Response to B. burgdorferi

The B. burgdorferi genome does not encode any recognizable toxin and instead causes infection by migrating through tissues, adhesion to host cells, and evasion of the immune response. Therefore, what actually causes the symptoms of Lyme disease such as erythema migrans, arthritis and neurology and cardiac problems, is the inflammatory response of the human innate immune system and the inability of both innate and adaptive immune responses to dispose of the bacteria.

Most often the body is capable of fighting off Lyme disease, especially with the help of antibiotic treatment. Generally, the body acts just as it normally would during a bacterial infection. In most patients, immune cells first encounter B. burgdorferi at the site of the tick bite. Complement-mediated lysis is possibly the first line of defense. The complement system works by expressing low levels of C3 is spontaneously hydrolyzed to C3a and C3b. Cb binds to certain surface motifs found on the surface of most cells. C3b then binds with Factor B and becomes C3bBb, which then binds to Factor P, which stabilizes C3bBb to C3 convertase and allows for the pathogen to be engulfed by macrophages. Host cells are able to protect themselves from opsonization because they contain membrane proteins, such as Factor H, which binds C3bBb, displaces it from the cell surface and allows it to be cleaved and inactivated by Factor I. B. burgdorferi has a way to get around this (Janeway 2000).

As part of the innate immune response, macrophages engulf and kill spirochetes by degrading them in intracellular compartments. Spirochetal lipoproteins and other spirochetal signals activate macrophages, leading to the production of a strong inflammatory response. Inflammatory cells within the erythema migran lesion produce primarily proinflammatory cytokines, including TNF-alpha and IFN-gamma. TNF-alpha is involved in inflammation and regulation of immune cells. IFN-gamma is a cytokine that is critical for innate and adaptive immunity against pathogens. It is a macrophage activator and is produced by CD4 and CD8 cytotoxic T lymphocyte effector T cells once antigen-specific immunity develops. Spirochetal lipoproteins also stimulate adaptive T cell-independent B cell responses (Steere et al. 2004)

B. burgdorferi-specific CD4+Th1 cells activate T cell-dependent B cell responses. Within days after the disease onset, most patients have and IgM antibody response to the outer surface protein C of the bacteria or the 41-kDa flagellar protein of the spirochete. This shows that both innate and adaptive cellular elements are active when fighting the infection. If the bacteria are not killed off early enough, it will spread through the body and the immune system continues to actively fight against the disease (Steere et al. 2004).

Overall schematic for how the immune system attacks B. burgdorferi
Host mechanism of spirochetal killing. Complement-mediated lysis of the organism may be the first line of host defense. Spirochetal lipoproteins and other spirochetal signals activate macrophages, leading to the production pro inflammatory cytokines, especially TNF-alpha and IL-1beta. Macrophages engulf spirochetes and degrade them in intracellular compartments. Spirochetal lipoproteins, which are B cell mitogens, also stimulate adaptive T cell-independant B cell responses. Humoral immune responses to nonlipidated spirochetal proteins are more likely to be T cell dependent. The primary role of B. burgdorferi-sepecific CD4+ Th1 cells is to prime T cell-dependent B cell responses, and antigen-specific CD8+ T cells may be a significant source of IFN-gamma. Antibpdy-mediated spirochetal killing occurs by complement fixation and opsonization. Steere et al. 2004.