MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.

Lyme Disease

Introduction EPIDEMIOLOGY Early in the 20th century, European physicians observed (LD), caused by infection with patients with a red, slowly expanding rash called erythema burgdorferi, is the most common vector-borne disease in the migrans (EM) (Figure 1) (1). They associated this rash with United States, accounting for more than 95% of all reported the bite of and postulated that EM was caused by a - vector-borne illnesses. From 2003-2005, 64,382 cases of borne bacterium. In the 1940s, a similar tick-borne illness Lyme disease were reported to the CDC from 46 states and was described that often began with EM and developed into the District of Columbia (2). However, a considerable level a multi-system illness. In 1969, a physician in Wisconsin of underreporting is associated with Lyme Disease and, diagnosed a patient with EM and successfully treated the therefore, the true number of infected individuals is probably individual with penicillin. In 1975, a women in Old Lyme, greater. Of the reported cases, 59,770 occurred within ten Connecticut contacted the state health authorities to ask why endemic states: Connecticut, Delaware, Maine, Maryland, so many children in her town had . She was alarmed Minnesota, New Jersey, New York, Pennsylvania, Rhode by the fact that 12 cases of juvenile arthritis, a relatively rare Island and Wisconsin (2). Within these endemic states, disease, had been diagnosed in a town with a population of the average annual rate of infection over this three year only 5,000. Subsequent investigations of these and similar period was 29.2 cases per 100,000 population (2). Three cases led to the discovery of Lyme disease and to the counties, New York’s Columbia and Dutchess counties and identification of the causative bacterium. Massachusetts’ Dukes County, were determined to have an incidence rate of 300 cases per 100,000 population (2). Evaluation of race and sex revealed a slightly higher incidence for males (54% of reported cases) and that 97% of the cases affect caucasians (2). Persons of all ages and both genders are equally susceptible, although the highest incidence of LD occurs in children ages 0-14 years and in persons 30 years of age and older (median, 28 years). LD has been reported in 49 states and the District of Columbia. Lyme borreliosis also occurs in temperate regions of the Northern Hemisphere in Europe (3), Scandinavia (4), the former Soviet Union (5), China (6) and Japan (7). In the United States, the infection is spreading and has caused focal epidemics, particularly in the northeastern United States (8-10). From 1985 to 1989, Figure 1: rash in Lyme borreliosis. the number of counties in New York State with documented I. scapularis ticks increased from 4 to 22, and the number of In 1982, spirochetes were identified in the midgut of the counties endemic to LD increased from 4 to 8 (11). During black-legged, adult deer tick, scapularis, and given the subsequent 10 years, I. scapularis ticks have spread to the name Borrelia burgdorferi. Finally, conclusive evidence 54 of the 62 counties in the state. In the US, the majority that B. burgdorferi caused Lyme disease came in 1984 when of affected individuals have had symptoms of the illness, spirochetes were cultured from the blood of patients with the whereas in Europe, the majority is asymptomatic (10, 11). rash EM and from the cerebrospinal fluid of a patient with Of 346 people who were studied in the highly endemic area meningoencephalitis and history of prior EM. The Centers for of Liso, Sweden, 41 (12%) had symptoms of the illness and Disease Control and Prevention (CDC) began surveillance 89 (26%) had evidence of subclinical infection (12). In a for Lyme disease in 1982. The Council of State and Territorial serosurvey of 950 Swiss orienteers, 26% had detectable IgG Epidemiologists (CSTE) designated Lyme disease as a antibodies to B. burgdorferi, but only 2% to 3% had a past nationally notifiable disease in January 1991. history of definite or probable LD (Figure 2) (1,13).

www.mdlab.com • 877 269 0090 (C) Adult female black-legged or deer tick (I. scapularis). As a member of the greater I. persulcatus group that also includes I. pacificus, I. ricinus, and I persulcatus, this tick is an important vector of several infectious agents that cause disease in humans, including Lyme borreliosis, (granulocytic) , Figure 2: Approximate geographic distributions of I. ovatus and . I. persulcatus (green with horizontal lines), I. pacificus (red with horizontal lines), ticks elsewhere in the world I. persulcatus (red), I. ricinus (green), I. scapularis (blue), R. evertsi may also transmit tick-borne flaviviruses capable of causing (yellow with horizontal lines), and R. pumilio (yellow with vertical , encephalomyelitis, and even hemorrhagic fevers lines). (including tick-borne encephalitis virus, louping ill virus, Russian spring-summer encephalitis virus, Powassan virus, Kyasanur Forest TICK BIOLOGY Disease virus, and Langat virus). Images courtesy of G. Maupin. Ticks evolved 94 million years ago from mites as parasites To accomplish a successful blood meal, a tick has to (i) attach of reptiles, birds, and mammals. More than 50 of to the host, (ii) impair local itching and pain responses to stay ticks can feed on humans (13, 14). Ticks belong to two undetected, (iii) prepare the tissue for blood extraction, (iv) taxonomic families, the soft ticks (Argasidae) and the hard prevent blood clotting, and (v) suppress the local immune ticks (Ixodidae). Humans are not the principal, but rather the and inflammatory responses (16). serendipitous host for most ticks (Figure 3) (1).

Figure 3: (A) Adult female Attachment: Upon finding a suitable host, the tick raises its lone star tick (Ameblyoma body at an angle to the skin surface and the chelicerae begin americanum). The lone star to cut the epidermis (17). Mouthpart lengths differ significantly, tick is broadly distributed throughout the southeastern with lengths in Dermacentor (dog ticks) extending to the quadrant of the United States, dermis-epidermis interface, while the mouthparts of Ixodes with ranges extending into New species extend well into the dermis (18). England and the Midwestern states. All three life cycle Cement: Salivary glands of Ixodid ticks generally secrete stages of A. americanum can an attachment cement, which is believed to serve as an bite humans. A. americanum adhesive and to seal the bite lesion bridging the cleft between is a recognized vector of mouthparts and host tissue (19). Attachment cement several pathogens that cause diseases in humans, including ehrlichioses caused by E. chaffeensis secretion begins within minutes after skin penetration and and E. ewingii, and has been implicated as a vector of southern tick- hardens into a tube surrounding the mouthparts. associated rash illness believed to be caused by Borrelia lonestari. Other pathogens or potential pathogens have also been detected Mechanics: Cutting action and mouthpart insertion produces in this tick, including various spotted fever group rickettsiae, an expanding hemorrhagic pool within the tissues, which is Francisella tularensis, and Coxiella burnetti, although the role of A. aided by anticoagulants, vasodilators, and aggregation americanum in the transmission of these agents to humans is not inhibitors in tick saliva (17). Mouthpart mechanical actions well characterized. (B) Adult alone are not sufficient to account for the lesion. The lesion female American dog tick (). is characterized by a large blood-filled cavity. Ixodid tick This tick is abundant in the blood feeding is not continuous (17). The first few days of southeastern United States hard tick feeding are referred to as the slow feeding phase, and coastal New England, which coincides with cuticle growth to accommodate the with limited distribution in blood meal. Next, the rapid feeding phase is characterized several midwestern states, by the uptake of very large quantities of blood. The increase southern Canada, and in size during the 7 to 10 day slow-feeding phase can be western coastal regions. 10-fold, with an additional 10-fold increase during the rapid- The tick is best known as the primary vector of Rocky feeding phase (20). This increase actually underestimates Mountain spotted fever in the amount the blood consumed, since fluid from the blood the eastern United States. meal is reintroduced into the host during salivation (20). MOLECULAR AND BIOLOGICAL Pain and Itch: Pain and/or itching are important host responses that alert an individual to the presence of a ASPECTS OF FEEDING tick, resulting in grooming behavior that removes the tick. Ticks evolved strategies to overcome host hemostasis, pain Bradykinin is a peripheral mediator of the sensations of and itch responses, , and immune defenses itching (17) and pain (22). I. scapularis saliva contains a by secreting saliva, which contains a complex array of metallodipeptidyl carboxypeptidase capable of degrading pharmacologically active molecules (Figure 4) (1). active bradykinin (23). Bradykinin also has roles in inflammation and increases vascular permeability (24). Histamine is one of several mediators that transmit the sensation of itching through peripheral sensory nerve endings (17). In addition, a combination of histamine and serotonin, a further mediator of itching, inhibits tick feeding (25). Histamine binding proteins are produced by the salivary glands of many tick species like Rhipicephalus sanguineus A (26) and I. scapularis (27).

Blood Coagulation and Tick Anticoagulants: Blood coagulation is a critical mechanism to prevent blood loss from the vasculature and poses a major obstacle for blood- feeding parasites, including ticks. Blood coagulation results B from activation of the extrinsic tissue factor and intrinsic pathways, which converge to form activated factor X (Xa) to initiate the common pathway of coagulation (28). Ticks have developed numerous redundant and complementary ways to inhibit coagulation. Ticks block host blood coagulation predominantly by targeting factor Xa and thrombin (29). Inhibitors of factor Xa have been reported from the saliva C or salivary glands of the following tick species: O. moubata, (30), R. appendiculatus, (31), and I. scapularis (32). I. scapularis saliva contains a further inhibitor of extrinsic factor X activation, Ixolaris, which is believed to utilize both factors X and Xa as scaffolds for the inhibition of activated factor VII/ D E tissue factor complex (29). Blocking the action of thrombin prevents conversion of fibrinogen to fibrin (27). Thrombin Figure 4: (A-D) Hard tick morphology. (A) Engorged Female A. inhibitors are produced by the salivary glands of I. holocyclus americanum. (B) Closeup of mouthparts of an I. scapularis nymph. (33) and I. ricinus (34). In addition to the inhibition of blood C, chelicerae; P, palps; H, hypostome. The mobile distal cheliceral coagulation, a successful blood feeding is highly dependent teeth reach beyond the hypostome. (C) During feeding, the palps on the inhibition of platelet aggregation. Platelet aggregation are splayed laterally (female D. andersoni tick). (D) A feeding can be inhibited by apyrase, which hydrolyzes both ATP and D. andersoni couple; the female tick is in front. (E) Tick feeding ADP to AMP and inorganic phosphate. Salivary apyrases are lesion. Adult I. scapularis female feeding on a sensitized rabbit. Predominantly mixed inflammatory infiltrate cells line the feeding present in I. scapularis (35) and many other blood feeding cavity. Discrete dermal swelling and dilated venules are found in arthropods (36). encoding putative metalloproteases the proximity. The mouthparts are anchored deep into the dermis, are present in the salivary glands of I. scapularis (27). Tick just reaching the cavity from which the tick feeds as indicated by the saliva metalloproteases could provide another means of imprint of the hypostome contours (trichrome stain). Photographs preventing platelet aggregation. In addition, the disruption of courtesy of S. Archibald and E. Denison. the likely contributes to maintaining the feeding lesion around tick mouthparts. Proteases and Protease Inhibitors: As mentioned earlier, the mechanical activity of the chelicerae will most certainly In addition to inhibiting platelet aggregation, prostaglandin E2 not entirely explain the relatively large blood-filled tissue is also a vasodilator. Prostaglandin E2 occurs in the salivary cavity that is characteristic of feeding ticks. A number of glands of I. scapularis (35). Vasodilation increases blood flow proteolytic activities and proteases have been characterized to the bite site, which is beneficial to engorging ticks. from various tick species. Multiple putative serine protease inhibitors have been identified in (21). www.mdlab.com • 877 269 0090 3 TICK INTERACTION WITH THE HOST wrapped around the inside the space between the cytoplasmic membrane and the outer membrane. Periodic IMMUNE SYSTEM contractions of these internal flagellae cause the outer The outcome of tick feeding and pathogen inoculation is surface to rotate, producing a corkscrew-type motility. governed by the interplay of the tick’s salivary pharmacologic This type of motility may be responsible for the ability of repertoire and the host’s innate and adaptive immune spirochetes to move out of the skin into the bloodstream and responses. Ticks target common pro-inflammatory pathways out of the bloodstream into tissues. Borrelia spp. do not have of activation and recruitment, such as bioactive amines, LPS in their outer membranes. Instead, the outer membrane chemokines and the . For example, contains a number of lipoproteins, some of which are adult D. reticulates and A. variegatum salivary gland extracts designated Osp, for outer surface proteins, A through F. Two inhibited human natural killer cells (NK), while I. ricinus had outer surface proteins, Osp A and Osp B, are encoded by the no effect (37, 38). IL-2, an important growth factor for NK same 50 kilobase linear and share 56% sequence cells and T cells, is blocked through a putative IL-2 binding homology (47) (Table 1). protein in the saliva of I. scapularis (39).

In addition, chemokines like IL-8, which recruit immune cells such as neutrophils, basophils, lymphocytes and monocytes to the site of tissue, insult are inhibited by activities in the saliva of several hard ticks (R. appendiculatus, D. reticulates and I. ricinus) (40).

The effects of tick saliva and tick feeding on T-cell cytokines have been studied extensively. The common denominator of these observations for several species is a reduced IFN-γ response and an upregulation of IL-4 (41, 42). Upregulation of IL-10 is not consistently observed (42, 43), but up regulation of IL-4 and IL-10, individually or combined, may account for the reduced IFN-γ induction (44). Figure 5: B. burgdorferi

Protein Features

The Organism • Up-regulated in maturing tick larva OspA • Down-regulated in nymphs during blood meals Based on genotyping of isolates from ticks, animals, and • Not expressed in mammals until late in infection • High homology (56%) with Osp A humans, three pathogenic groups of Borrelia burgdorferi OspB • ospA in operon with ospB gene on linear plasmid

(senso lato) have been identified. In the United States, the • Encoded by a 26 kb circular plasmid OspC • Up-regulated as the spirochete moves from the mid-gut to the sole cause of human infection is group one, B. burgdorferi salivary gland (senso stricto). Group one can also be found in Europe OspD (Erps) • Produced early during mammalian infection and Asia. Although all three groups are found in Europe, OspE-OspF • Immunogenic in some patients DbpA and DbpB (decorin • Binds mammalian protein decorin the predominant groups are group 2 (B. garinii) and group 3 binding protein) • Produced by in a mammal • Surface protein that varies antigenically, similar to variable membrane (B. afzelii). Only groups 2 and 3 have been associated with VlsE proteins found in B. hermsii (the causative agent of ) Lyme disease in Asia. Although closely related, the disease patterns observed in humans differ greatly depending upon Table 1: Features of major surface proteins of B. burgdorferi. the particular Borrelia species involved. The Borrelia Borrelia species are spirochetes, spiral-shaped organisms that have a gram-negative cell wall architecture consisting Borrelia species have linear as well as the of two membranes, between which is sandwiched the conventional circular plasmids. Linear plasmids were first layer (Figure 5) (45). The Borrelia spp. are discovered by scientists studying B. burgdorferi, a feature longer and more loosely coiled than other spirochetes. Of originally thought to be unique to this . In recent years, the Borrelia spp., B. burgdorferi is the longest (20 to 30 mm) however, linear plasmids and have been and narrowest (0.2 to 0.3 mm) and has fewer flagellae (8- found in a number of other bacterial groups. The two strands 12, 46). The bacteria are actively motile, but the flagellae do of the double stranded linear plasmids are covalently closed not extend outward from the surface of the bacterium like at the ends and have sequences similar to telomeres found those of most gram-negative bacteria. Rather, the flagellae on eukaryotic chromosomes. The mechanisms by which the are embedded into the two ends of the spirochete and are linear plasmids of spirochetes replicate is still unknown. The complete genome of a prototypic B. burgdorferi strain Initially, the immune response in Lyme disease seems to be (B31) was sequenced (48). The total genome is small suppressed (57), which may be an important mechanism (approximately 1.5 megabases) and includes a linear in allowing the spirochete to disseminate. Within days to of 950 kilobases along with 9 circular and 12 weeks, peripheral blood mononuclear cells begin to have linear plasmids. The plasmid comprises approximately 40% heightened responsiveness to B. burgdorferi antigens and of the bacterial genome. The organism has a minimal number mitogens (58, 59). In vitro, B. burgdorferi is a potent inducer of of proteins with biosynthetic activity and therefore depends pro-inflammatory cytokines, including tumor necrosis factor- on the host for much of its nutritional requirements. Analysis γ and interleukin-1b from peripheral blood mononuclear cells of B. burgdorferi plasmid DNA content reveals that extensive (60). recombination occurs between plasmids leading to genetic rearrangements. What role this variability of plasmid-borne The antibody response to B. burgdorferi develops slowly. genes plays in adaptation of the bacteria to its various hosts The specific IgM response spikes between the third and remains to be determined. the sixth week of infection (61) and often is associated with polyclonal activation of B cells, including elevated total serum Analysis of the Borrelia genome reveals the absence of any IgM levels (62), circulating immune complexes (63), and genes encoding cytochromes or tricarboxylic acid (TCA) cryoglobulins (64). Membrane lipoproteins, including OspA, cycle enzymes, which is consistent with the observation that are mitogenic for B cells (65). The specific IgG response although B. burgdorferi can grow in the presence of oxygen, develops gradually over months to an increasing array of it seems not to need oxygen for growth. Because the bacteria spirochetal polypeptides (66) and non-protein antigens lack a TCA cycle, they can not make any of the amino (67). Histologically, all affected tissues show an infiltration acids whose synthesis requires TCA cycle intermediates. of lymphocytes and plasma cells (68). Some degree of Given this, it appears odd at first that there were few genes vascular damage, including mild vasculitis or hypervascular encoding amino acid transport proteins. This is consistent, occlusion, may be seen in multiple sites, suggesting that however, with the observation that the bacteria preferentially the spirochete may have been in or around blood vessels. take up peptides rather than individual amino acids. Despite an immune response, B. burgdorferi may survive for years in untreated patients in certain niches within the joints, nervous system, or skin; it is not yet known how it is able to sequester itself in these sites. PATHOGENESIS To maintain its complex enzootic cycle, B. burgdorferi must adapt to two markedly different environments, the tick and CLINICAL SIGNIFICANCE the mammalian host. For example, in the mid-gut of the tick, Lyme disease occurs in stages, with remissions, the spirochete expresses OspA and OspB (48). When the exacerbations and different clinical manifestations at blood meal is taken, OspC is up-regulated as the organism each stage. Early infection consists of stage 1 (localized migrates to the tick salivary gland and the mammalian host EM) (Figures 1,6) (1), followed by stage 2 (disseminated (49). After injection of the spirochete by the tick and an infection), and finally stage 3 (late infection or persistent incubation period of 3 to 32 days, the spirochete usually infection). Stage 3 usually begins months or years after first multiplies locally in the skin at the site of the tick bite. disease onset, sometimes following long periods of latent Several days later, the spirochete begins to spread in the infection (69). In an individual patient, however, the infection skin and within days to weeks it may disseminate further. is highly variable, ranging from brief involvement in only one Bacterial spread within the host is probably facilitated by system, to chronic multi-system involvement of the skin, the spirochete’s ability to bind human plasminogen and nerves, and joints for a period of years. urokinase-type plasminogen activator to its surface (50, 51). Plasmin, the active proteinase form of plasminogen, can promote the tissue invasion capability of B. burgdorferi. To date, two binding and cellular entry mechanisms have been identified. First, the spirochete attaches to members of the integrin family of receptors and the vitronectin and fibronectin receptors (52, 53). A second pathway for cell attachment is mediated by host cell sugars, particularly glycosaminoglycans (54). In in vitro systems, intracellular localization of B. burgdorferi has been demonstrated within human endothelial cells, macrophages, and fibroblasts (55, 56, 57). Figure 6: Erythema migrans rash in Lyme borreliosis www.mdlab.com • 877.269.0090 5 Early Infection: Stage I (Localized pleocytosis without headache, and sometimes cranial (74). Within several weeks after the onset of illness, about Infection) 5% of untreated patients develop cardiac involvement (75) The first phase, or the skin phase, follows inoculation of the with the most common abnormality as fluctuating degrees of spirochete into the skin by a tick during its blood meal. The (75, 76). However, some patients have Ixodes tick that transmits Lyme disease to humans is a pool evidence of electrocardiographic changes compatible with feeder in that it does not inject the bacteria directly into the acute myopericarditis (77). blood stream but creates a lesion into which blood flows. (Figure 4E) Thus, the bacteria are originally introduced During this stage, musculoskeletal pain is common. The into the lower layers of skin. EM, which occurs at the site of typical pattern is one of migratory pain in joints, tendons, the bite, usually begins as a red macule or papule. In one bursae, muscle or bones, often without joint swelling. study, only 31% of patients (N=314) recalled a tick bite at In addition, a few patients have been described with the skin site where EM developed 3 to 32 days later (70). osteomyelitis and myositis (78, 79). Conjunctivitis is the The centers of early lesions sometimes become intensely most common eye abnormality in Lyme disease, but deeper erythematous and indurated, vascular, or necrotic. In some tissues in the eye may be affected as well (70). instances, migrating lesions remain an even intense red, have several red rings which are found within the outside ring, or the central area turns blue before it clears. It has Late Infection: Stage III (Persistent been documented that as many as 40% of patients do not Infection) exhibit this characteristic skin manifestation. This initial skin phase may give the spirochete some protection from the The third stage of the infection begins when the spirochete neutrophils that patrol the blood stream. The ability of the enters tissue throughout the body. At that point, the bacteria spirochete to move away from the region may also play a become more difficult to find and may enter a quiescent protective role. phase with lowered metabolic activity, which makes them less likely to attract the cells of the host’s defense systems. Months after the onset of the illness, about 60% of patients Early Infection: Stage II (Disseminated begin to experience intermittent attacks of joint pain and swelling, primarily in large joints, especially the knee (80). Infection) Although the spirochete has been cultured from the joint fluid The spirochete next invades the bloodstream, leading to of only two patients with Lyme arthritis (69), B. burgdorferi further dissemination throughout the body. Within days DNA may be detected by polymerase chain reaction (PCR) after the onset of the initial EM skin lesion, patients may in the synovial tissue or joint fluid of most patients (81, 82).B. experience multiple annular secondary lesions (70, 71). burgdorferi–specific T cells, reactive with multiple spirochetal During this period, some patients develop macular rash, polypeptides, are concentrated in joint fluid (59, 83). These conjunctivitis, or, rarely, diffuse urticaria. EM and secondary T cells secrete primarily the pro-inflammatory Th1 cytokine lesions usually fade within 3 to 4 weeks (range, 1 day to IFN-γ. This response is dominant in synovium, a pattern 14 months). EM is often accompanied by malaise, fatigue, that leads to a delayed hypersensitivity response (84). From headache, fever, chills, generalized achiness, and regional months to years after disease onset, sometimes following lymphadenopathy (70, 71). In addition, patients may have long periods of latent infection, patients may develop chronic evidence of meningeal irritation, mild encephalopathy, neurologic manifestations of the disorder (84, 85). The most migratory musculoskeletal pain, sore throat and cough. common form of chronic central nervous system involvement Except for fatigue and lethargy, which are often constant, is a sub-acute encephalopathy affecting memory, mood, or the early sign and symptoms are typically intermittent and sleep (87, 88). Patients with these symptoms often have changing. After several weeks to months, however, 15% CSF abnormalities, including increased CSF protein levels, of untreated patients in the United States develop frank evidence of B. burgdorferi antibodies, or both (87, 88). neurologic abnormalities, including , encephalitis, and cranial neuritis, including bilateral facial palsy (72). The usual pattern consists of fluctuating symptoms of meningitis Borrelia garinii with superimposed cranial neuritis, particularly facial palsy. Borrelia garinii is transmitted by the ticks Ixodes ricinus and On examination, such patients usually have neck stiffness. Ixodes persulcatus and appears to be the most neurotropic Facial palsy, or Bell’s palsy, frequently occurs alone and of the three Borrelia species. Borrelia garinii may cause may be the presenting manifestation of the disease (73). an exceptionally wide range of neurologic abnormalities In Europe, the most common manifestation is Bannwarth’s including borrelial encephalomyelitis, a multiple sclerosis–like syndrome, which includes neuritic pain, lymphocytic illness, which is a severe neurologic disorder characterized by spastic parapareses, ataxia, cognitive impairment, DIAGNOSIS bladder dysfunction, and cranial neuropathy, accompanied Indirect (Antibody Detection Tests) by antibody production to B. burgdorferi. Antibody tests are indirect, in that they measure exposure to a pathogen. A positive test is a function of the host immunological response to a foreign antigen, B. burgdorferi Borrelia afzelii is transmitted by the ticks Ixodes ricinus in the case of Lyme disease. Direct tests are those that and Ixodes persulcatus. In Europe and Asia, B. afzelii may detect the entire bacterium (usually by culture or staining persist in the skin for decades resulting in acrodermatitis techniques) or parts of the bacterium such as cell wall chronica atrophicans (ACA) (Figure 7) (1). ACA occurs proteins, carbohydrates, intracellular proteins, or nucleic during the third stage, or late stage, of European Lyme acids such as DNA or RNA. Both indirect (serology) and Borreliosis, and is a skin condition that occurs primarily on direct tests (PCR) are available for the accurate, sensitive, sun-exposed surfaces of distal extremities beginning with and specific laboratory detection of Lyme disease. Because an inflammatory stage with bluish-red discoloration and of the complex nature of the disease, no single test may be cutaneous swelling and concluding several months or years sufficient for all clinical situations. later with an atrophic phase. Sclerotic skin plaques may also develop. The frequency of ACA is about 1-10% of all Medical Diagnostic Laboratories (MDL) offers three serology European patients with Lyme Borreliosis, varying according tests for the detection of antibodies to Borrelia burgdorferi, to the region of the population sampled. ACA is probably the the causative agent of Lyme disease (LD): most common late and chronic manifestation of borreliosis in European patients with Lyme disease. Because the 1. First step Enzyme-Linked Immunosorbent Assay clinical diagnosis of ACA is much more difficult than that of (ELISA) screening test EM or Borrelia lymphocytoma (BL), the condition is often 2. Supplemental Western immunoblot (IgG, IgM) underdiagnosed, and, in fact, the ratio of EM cases to ACA 3. C6 Lyme peptide assay (C6LPA) cases may be higher. A. The Enzyme-Linked Immunosorbent Assay (ELISA) screening test Enzyme-Linked Immunosorbent Assays rely on an antigen’s ability to adsorb to a solid phase. When a serum specimen is introduced to this system, any antigen-specific antibodies which may be present in the patient serum will bind to the antigen pre-adhered to the solid support such as a microwell plate. Once excess antibody is washed from the surface of the solid phase, goat anti-human IgG/IgM conjugated with horseradish peroxidase is then introduced. It will bind to any antigen-antibody complexes which are adhered to the solid phase, forming a sandwich. Once excess conjugate is washed from the solid phase, Chromagen/Substrate Tetramethylbenzidine (TMB) is then introduced. If specific antibodies are present because they formed a sandwich with the antigen-antibody complexes, the solution will turn blue.

Once the stop solution, 1N H2SO4, is added, the solution turns yellow and is measured using a spectrophotometer. Interpretation of the resulting reading is indicative of the concentration of antibody in the patient serum specimen (Figure 8) (88).

Figure 8: ELISA Assay Figure 7: Acrodermatitis chronica atrophicans, a late skin manifestation of B. afzelii infection, shown affecting the legs of an elderly woman where the skin has become thinned and atrophic. Image courtesy of R. Muellegger. www.mdlab.com • 877 269 0090 7 The ELISA should only be ordered for patients who have test is positive or negative. In the Western blot sample signs and symptoms consistent with Lyme disease. Equivocal illustrated in (Figure 9), the top strip is from a patient positive or positive ELISA tests are recommended by the CDC to for IgG and the bottom strip is the band locator strip which be followed with a supplemental Western blot. However, is used for comparative purposes to help properly identify some clinicians may wish to use a Western blot test as a band location. companion test to the ELISA. While positive first-step ELISA results supplemented with a positive second-step Western blot can be used to support a diagnosis of Lyme disease, negative results of either an ELISA or Western blot should not be used to exclude Lyme disease.

Early antibody responses are often due to the gp41 flagellar Figure 9: An IgG positive patient Western blot strip (top) in antigen. Due to its cross-reactive components, a false comparison to a band locator strip (bottom). positive reaction will often occur with patients or other patients with spirochetal diseases, such as The CDC recommends an interpretation of Western or periodontal disease, since these treponemes also have blot based on the work of Dressler et al. (90). The CDC similar gp41 flagellar antigens. This degree of cross- CRITERIA includes: reactivity results in low specificity of the ELISA assay and it is therefore recommended that positive or equivocal first-step ELISA results be supplemented by a second-step assay. IgM Significant Bands:

Reactive: At least two of the following three bands must be B. Western immunoblot (IgG, IgM) B. burgdorferi present: 23, 39, 41. antibodies Early tests for the laboratory diagnosis of Lyme disease IgG Significant Bands: suffered from a lack of both sensitivity and specificity. Reactive: At least five of the following bands must be Enzyme linked immunoassays (ELISA) used whole cell present: 18, 23, 28, 30, 39, 41, 45, 58, 66, 93. lysate preparations of B. burgdorferi as capture antigen and were particularly susceptible to false positive results because of cross-reactive antibodies present in the patient sample. In Non-Reactive: Fewer than two bands are present. 1989, the Centers for Disease Control and Prevention (CDC) * Note that the 31 and 34 kDa B. burgdorferi specific bands recommended that all indeterminate and positive ELISA tests represent outer surface proteins A and B, respectively, have be confirmed by Western blot (88). Subsequently, tests have been excluded from the IgG criterion. Note also, that there become available which are both more sensitive and specific is no indeterminate or equivocal category— five bands are interpreted as positive while four bands constitute a negative but the majority of clinical laboratories still use a whole cell result. Included in the IgG interpretive criterion is the 41 kDa lysate ELISA screening test with a supplemental Western band observed in 65% of the normal population. blot test performed on indeterminate and positive screening results. Tilton et al. (91) and others have proposed the following ALTERNATE CRITERIA: The Western blot is an antibody test which is capable of differentiating the antibody response to the variety IgM significant bands: of B. burgdorferi antigens. The antigens are separated electrophoretically and then transferred, or “blotted”, to a Reactive: Two or more of the following four bands must be nitrocellulose strip. The strips are exposed to the patient present: 23, 39, 41, 83/93. sample containing antibodies that are both specific to B. burgdorferi as well as other infectious agents. The presence IgG significant bands: of colored bands on the strips is indicative of an antigen/ Reactive: Three or more of the following bands must be antibody reaction. The molecular weight of the antigen can be present: 20, 23, 31, 34, 35, 39, 83/93. determined by its location on the strip; hence the numerical designation of the antigen, OspA (31 kDa), OspB (34 kDa), and so on. Because many of the antigens that appear on the Equivocal: One of the following bands must be present: 23, 31, 34, 37, 39, 41, 83/93 Western blot strip are shared by other organisms in addition to B. burgdorferi, the interpretation of the blot becomes critically important in determining whether the Western blot Non-reactive: No Lyme-specific bands present. The major differences are: complexity of a whole cell lysate, a high background is often present and false positives are therefore a problem. IgM – This interpretive criterion is similar to the CDC except This is the basis for the lack of specificity inherent to most that 83/93 has been included as a significant IgM band enzyme immunoassay (EIA) kits and thereby necessitates that is specific to B. burgdorferi. The Alternate Criteria also confirmatory immunoblot tests to distinguish Lyme-specific incorporates an “Equivocal” category that includes late from non-specific antibodies. appearing antibodies specific for the 31 kDa OspA and 34 kDa OspB antigens. Yet another problem with whole cell ELISAs is the presence of antibodies to OspA that are vaccine induced. Separating IgG – The Alternate Criterion is based on both the number of the vaccine response from the natural host response to bands and the significance of the antibodies detected. For infection becomes very difficult with whole cell ELISAs. example, Osp A (31 kDa) and B (34 kDa) are important bands often seen in late stages of Lyme disease. The alternate Single or multiple peptide tests use only antigens that may criteria also includes an “Equivocal” category which indicates be specific for B. burgdorferi. These include tests using that although there are insufficient antibody bands present recombinant OspA, OspB, and flagellar proteins (91), for the blot to be reactive, there is significant immunologic recombinant OspC, p39 and flagellar proteins, p83 antigen, activity observed which may be related to Lyme disease. the hook protein of B. burgdorferi (FlgE), p37, and the C6 Lyme peptide recombinant or synthetic antigen (93, 94). To determine the effect of reporting results using the CDC The single peptide Lyme C6 EIA for Lyme disease has vs. the alternate criteria (92), commercially available panels been approved by the Food and Drug Administration (FDA). (BBI Diagnostics, CDC) of characterized serum samples The Borrelia burgdorferi C6 peptide antibody test (C6 LPA) were used. The results indicated that the use of the Alternate identifies antibodies to a newly discovered, conserved Criteria markedly increased the sensitivity of the Western peptide called C6, which is a component of the variable blot while only decreasing the specificity slightly. surface antigen of B. burgdorferi. The C6 LPA is important because it detects both IgM and IgG antibodies in patients This raises the question at to whether a Western blot with LD but not in vaccinees. should be more sensitive or as specific. If the Western blot is to be used solely for confirmation of ELISA results, then The C6 LPA, like some other peptide assays (the exception specificity may be preferred over sensitivity. However, a being the OspA EIA), can effectively discriminate vaccinated specific Western blot with low sensitivity may invalidate a from unvaccinated patients with Lyme disease. One sensitive and specific ELISA. A Western blot that is both hundred percent of vaccinees tested had a negative Lyme highly sensitive and specific is desirable for a two-tiered C6 EIA. The C6 LPA FDA submission reported sensitivities test scheme. However, despite the recommendations of for acute, convalescent and late stage disease of 74%, 90%, the CDC for two-tiered testing, many physicians who treat and 100%, respectively (93, 94). More recent unpublished patients with Lyme disease do not accept ELISA as a very data (99) suggest that the sensitivity of the C6 LPA in chronic sensitive screening test and, therefore, request both ELISA Lyme disease may not be as high as originally anticipated and Western blot be performed on their patients. but no worse, and probably better, than any other available ELISA (99). These levels of sensitivity and the much improved specificity (99%) suggests that the C6 LPA might ** Please note that BOTH the Alternate and the CDC become the test of choice for screening purposes. Serum criteria will appear on all Lyme Western blot result reports specimens from patients with early are 95% from Medical Diagnostic Laboratories. Occasionally, this positive. The early C6 response to infection includes both will cause differences in results; your alternate report IgM and IgG production and may appear soon after a tick will also include an equivocal category. bite (93, 94). Patients with over a dozen different diseases including systemic lupus erythematosus, non-Lyme arthritis, C. The C6 Lyme Peptide Antibody Test syphilis, other spirochetal diseases, and other autoimmune diseases were uniformly C6 negative. Antibodies produced Commercially available Lyme disease antibody kits, as well by challenge with B. burgdorferi, B. afzelli, and B. garinii as most research level Lyme disease kits, use a whole cell can all be detected with the C6 LPA. Although the latter two lysate as the capture antigen. The capture antigen is used strains are not indigenous to the United States, both are to coat a solid support such as a microwell plate and binds prevalent in Europe. specific antibodies that may be present in the serum sample. These lysates contain multiple epitopes, not all of which All current positive serological screening tests should be are specific for B. burgdorferi. Because of the immunologic confirmed by Western blot. With more clinical experience, www.mdlab.com • 877 269 0090 9 the high specificity of a positive C6 LPA may eliminate the to traditional PCR and has improved the specificity and need for confirmatory testing (95). The immune response to sensitivity of diagnostic testing of this type of testing. Like the OspA vaccine has been reported to induce antibodies traditional PCR, real-time assays utilize sequence-specific that bind to multiple proteins of B. burgdorferi (95). Sera primers that allow for the highly specific amplification of DNA from vaccinated people show multiple low molecular weight sequences from minute amounts of target material. What bands in addition to a broad band at 31 kDa on Western makes this technology superior, however, is the incorporation blot. The utility of a Western blot in a vaccinated person is of a sequence-specific fluorescent reporter probe that allows questionable. Until recently, there has been no “test of cure” for an accurate quantitation of the reaction following each for Lyme disease and the clinical determination of a “cure” is amplification cycle. very controversial. Whether persistent symptoms (persistent Lyme disease, post-Lyme syndrome, ) The limitations associated with conventional Lyme testing are after adequate therapy are due to active B. burgdorferi especially evident when taking into consideration infections infection, existing tissue injury, a post-infectious syndrome with Borrelia lonestari, a clinically similar, yet genomically or a condition unrelated to Lyme disease is not well defined. distinct, pathogenic strain. In this instance, serum samples Routine ELISAs do not correlate with the disappearance or from individuals determined to be infected with B. lonestari reappearance of symptoms. In contrast, C6 LPA antibodies were unable to recognize B. burgdorferi antigens, rendering diminished by a factor of >4 in successfully treated patients conventional ELISAs useless (96, 97). Despite the fact that and <4 in patients who did not respond to appropriate therapy most of these organisms are separated geographically for Lyme disease. Control tests using a whole cell ELISA or a from one another and, as such, infection with these agents p39 recombinant EIA were either unchanged or showed no would be more presumptive, the high rate of travel within significant change in titer as a result of treatment (93, 94). The the Unites States and to Europe means that none of these results suggest that quantitative C6 LPAs administered over pathogens should be discounted when a patient presents a period of time may be useful for monitoring a response to with symptoms typical of Lyme disease. The relevance of therapy as well as to determine whether the spirochete has such speciation tests is further supported by the report of been eliminated. Finally, the question of active vs. inactive an individual from Westchester County, New York who Lyme disease may be resolved because a positive C6 LPA was infected with B. lonestari during a trip to Maryland and suggests active infection and a negative test, even with a North Carolina (98). Therefore, in instances where patient positive routine ELISA, suggests inactive infection or no symptoms are suggestive of Lyme disease yet conventional infection at all, except in very early acute infections (93, 94). Lyme tests return negative or ambiguous results, direct screening via PCR-based technology should be employed. Direct Tests (Molecular Detection via Borrelia lonestari Real-Time PCR) Since the mid-1980’s, physicians in non-Lyme disease DNA detection by Real-Time PCR is available from MDL for endemic areas of the south eastern and south central portion whole blood, serum, urine, CSF, and synovial fluid samples. of the United States have described a Lyme disease-like Four individual assays were developed to differentiate illness. Two to twelve days following the bite of the Lonestar between the three B. burgdorferi sensu lato genospecies tick, , patients would describe an known to cause Lyme disease in humans, B. burgdorferi erythema migrans (EM)-like rash and mild flu-like symptoms sensu stricto, B. garinii and B. afzelii, as well as the clinically including fever. This condition is often referred to as southern related, but genomically distinct, B. lonestari. tick-associated rash illness (STARI), Master’s disease, or southern Lyme disease. 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