Lyme Disease

MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.

www.mdlab.com • 877 269 0090

IntroductIonEarly in the 20th century, European physicians observed

patients with a red, slowly expanding rash called erythema

migrans (EM) (Figure 1) (1). They associated this rash with

the bite of ticks and postulated that EM was caused by a tick-

borne bacterium. In the 1940s, a similar tick-borne illness

was described that often began with EM and developed into

a multi-system illness. In 1969, a physician in Wisconsin

diagnosed a patient with EM and successfully treated the

individual with penicillin. In 1975, a women in Old Lyme,

Connecticut contacted the state health authorities to ask why

so many children in her town had arthritis. She was alarmed

by the fact that 12 cases of juvenile arthritis, a relatively rare

disease, had been diagnosed in a town with a population of

only 5,000. Subsequent investigations of these and similar

cases led to the discovery of Lyme disease and to the

identification of the causative bacterium.

EPIdEMIoLoGYLyme disease (LD), caused by infection with Borrelia

burgdorferi, is the most common vector-borne disease in the

United States, accounting for more than 95% of all reported

vector-borne illnesses. From 2003-2005, 64,382 cases of

Lyme disease were reported to the CDC from 46 states and

the District of Columbia (2). However, a considerable level

of underreporting is associated with Lyme Disease and,

therefore, the true number of infected individuals is probably

greater. Of the reported cases, 59,770 occurred within ten

endemic states: Connecticut, Delaware, Maine, Maryland,

Minnesota, New Jersey, New York, Pennsylvania, Rhode

Island and Wisconsin (2). Within these endemic states,

the average annual rate of infection over this three year

period was 29.2 cases per 100,000 population (2). Three

counties, New York’s Columbia and Dutchess counties and

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,

the number of counties in New York State with documented

I. scapularis ticks increased from 4 to 22, and the number of

counties endemic to LD increased from 4 to 8 (11). During

the subsequent 10 years, I. scapularis ticks have spread to

54 of the 62 counties in the state. In the US, the majority

of affected individuals have had symptoms of the illness,

whereas in Europe, the majority is asymptomatic (10, 11).

Of 346 people who were studied in the highly endemic area

of Liso, Sweden, 41 (12%) had symptoms of the illness and

89 (26%) had evidence of subclinical infection (12). In a

serosurvey of 950 Swiss orienteers, 26% had detectable IgG

antibodies to B. burgdorferi, but only 2% to 3% had a past

history of definite or probable LD (Figure 2) (1,13).

Figure 1: Erythema migrans rash in Lyme borreliosis.

In 1982, spirochetes were identified in the midgut of the

black-legged, adult deer tick, Ixodes scapularis, and given

the name Borrelia burgdorferi. Finally, conclusive evidence

that B. burgdorferi caused Lyme disease came in 1984 when

spirochetes were cultured from the blood of patients with the

rash EM and from the cerebrospinal fluid of a patient with

meningoencephalitis and history of prior EM. The Centers for

Disease Control and Prevention (CDC) began surveillance

for Lyme disease in 1982. The Council of State and Territorial

Epidemiologists (CSTE) designated Lyme disease as a

nationally notifiable disease in January 1991.

tIcK BIoLoGY Ticks evolved 94 million years ago from mites as parasites

of reptiles, birds, and mammals. More than 50 species of

ticks can feed on humans (13, 14). Ticks belong to two

taxonomic families, the soft ticks (Argasidae) and the hard

ticks (Ixodidae). Humans are not the principal, but rather the

serendipitous host for most ticks (Figure 3) (1).

Attachment: Upon finding a suitable host, the tick raises its

body at an angle to the skin surface and the chelicerae begin

to cut the epidermis (17). Mouthpart lengths differ significantly,

with lengths in Dermacentor (dog ticks) extending to the

dermis-epidermis interface, while the mouthparts of Ixodes

species extend well into the dermis (18).

cement: Salivary glands of Ixodid ticks generally secrete

an attachment cement, which is believed to serve as an

adhesive and to seal the bite lesion bridging the cleft between

mouthparts and host tissue (19). Attachment cement

secretion begins within minutes after skin penetration and

hardens into a tube surrounding the mouthparts.

Mechanics: Cutting action and mouthpart insertion produces

an expanding hemorrhagic pool within the tissues, which is

aided by anticoagulants, vasodilators, and platelet aggregation

inhibitors in tick saliva (17). Mouthpart mechanical actions

alone are not sufficient to account for the lesion. The lesion

is characterized by a large blood-filled cavity. Ixodid tick

blood feeding is not continuous (17). The first few days of

hard tick feeding are referred to as the slow feeding phase,

which coincides with cuticle growth to accommodate the

blood meal. Next, the rapid feeding phase is characterized

by the uptake of very large quantities of blood. The increase

in size during the 7 to 10 day slow-feeding phase can be

10-fold, with an additional 10-fold increase during the rapid-

feeding phase (20). This increase actually underestimates

the amount the blood consumed, since fluid from the blood

meal is reintroduced into the host during salivation (20).

Figure 2: Approximate geographic distributions of I. ovatus

(green with horizontal lines), I. pacificus (red with horizontal lines),

I. persulcatus (red), I. ricinus (green), I. scapularis (blue), R. evertsi

(yellow with horizontal lines), and R. pumilio (yellow with vertical

lines).

Figure 3: (A) Adult female

lone star tick (Ameblyoma

americanum). The lone star

tick is broadly distributed

throughout the southeastern

quadrant of the United States,

with ranges extending into New

England and the Midwestern

states. All three life cycle

stages of A. americanum can

bite humans. A. americanum

is a recognized vector of

several pathogens that cause

diseases in humans, including ehrlichioses caused by E. chaffeensis

and E. ewingii, and has been implicated as a vector of southern tick-

associated rash illness believed to be caused by Borrelia lonestari.

Other pathogens or potential pathogens have also been detected

in this tick, including various spotted fever group rickettsiae,

Francisella tularensis, and Coxiella burnetti, although the role of A.

americanum in the transmission of these agents to humans is not

well characterized. (B) Adult

female American dog tick

(Dermacentor variabilis).

This tick is abundant in the

southeastern United States

and coastal New England,

with limited distribution in

several midwestern states,

southern Canada, and

western coastal regions.

The tick is best known as

the primary vector of Rocky

Mountain spotted fever in

the eastern United States.

(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) anaplasmosis,

and babesiosis. I. persulcatus

ticks elsewhere in the world

may also transmit tick-borne flaviviruses capable of causing

encephalitis, encephalomyelitis, and even hemorrhagic fevers

(including tick-borne encephalitis virus, louping ill virus, Russian

spring-summer encephalitis virus, Powassan virus, Kyasanur Forest

Disease virus, and Langat virus). Images courtesy of G. Maupin.

To accomplish a successful blood meal, a tick has to (i) attach

to the host, (ii) impair local itching and pain responses to stay

undetected, (iii) prepare the tissue for blood extraction, (iv)

prevent blood clotting, and (v) suppress the local immune

and inflammatory responses (16).

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MoLEcuLAr And BIoLoGIcAL ASPEctS oF FEEdInGTicks evolved strategies to overcome host hemostasis, pain

and itch responses, inflammation, and immune defenses

by secreting saliva, which contains a complex array of

pharmacologically active molecules (Figure 4) (1).

Figure 4: (A-D) Hard tick morphology. (A) Engorged Female A.

americanum. (B) Closeup of mouthparts of an I. scapularis nymph.

C, chelicerae; P, palps; H, hypostome. The mobile distal cheliceral

teeth reach beyond the hypostome. (C) During feeding, the palps

are splayed laterally (female D. andersoni tick). (D) A feeding

D. andersoni couple; the female tick is in front. (E) Tick feeding

lesion. Adult I. scapularis female feeding on a sensitized rabbit.

Predominantly mixed inflammatory infiltrate cells line the feeding

cavity. Discrete dermal swelling and dilated venules are found in

the proximity. The mouthparts are anchored deep into the dermis,

just reaching the cavity from which the tick feeds as indicated by the

imprint of the hypostome contours (trichrome stain). Photographs

courtesy of S. Archibald and E. Denison.

Proteases and Protease Inhibitors: As mentioned earlier,

the mechanical activity of the chelicerae will most certainly

not entirely explain the relatively large blood-filled tissue

cavity that is characteristic of feeding ticks. A number of

proteolytic activities and proteases have been characterized

from various tick species. Multiple putative serine protease

inhibitors have been identified in Ixodes ricinus (21).

Pain and Itch: Pain and/or itching are important host

responses that alert an individual to the presence of a

tick, resulting in grooming behavior that removes the tick.

Bradykinin is a peripheral mediator of the sensations of

itching (17) and pain (22). I. scapularis saliva contains a

metallodipeptidyl carboxypeptidase capable of degrading

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

(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

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

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/

tissue factor complex (29). Blocking the action of thrombin

prevents conversion of fibrinogen to fibrin (27). Thrombin

inhibitors are produced by the salivary glands of I. holocyclus

(33) and I. ricinus (34). In addition to the inhibition of blood

coagulation, a successful blood feeding is highly dependent

on the inhibition of platelet aggregation. Platelet aggregation

can be inhibited by apyrase, which hydrolyzes both ATP and

ADP to AMP and inorganic phosphate. Salivary apyrases are

present in I. scapularis (35) and many other blood feeding

arthropods (36). Genes encoding putative metalloproteases

are present in the salivary glands of I. scapularis (27). Tick

saliva metalloproteases could provide another means of

preventing platelet aggregation. In addition, the disruption of

the extracellular matrix likely contributes to maintaining the

feeding lesion around tick mouthparts.

In addition to inhibiting platelet aggregation, prostaglandin E2

is also a vasodilator. Prostaglandin E2 occurs in the salivary

glands of I. scapularis (35). Vasodilation increases blood flow

to the bite site, which is beneficial to engorging ticks.

A

B

C

D E

Protein Features

OspA• Up-regulated in maturing tick larva • Down-regulated in nymphs during blood meals• Not expressed in mammals until late in infection

OspB• High homology (56%) with Osp A • ospA gene in operon with ospB gene on linear plasmid

OspC• Encoded by a 26 kb circular plasmid • Up-regulated as the spirochete moves from the mid-gut to the salivary gland

OspD (Erps) • Produced early during mammalian infection

OspE-OspF • Immunogenic in some patients

DbpA and DbpB (decorin

binding protein)

• Binds mammalian protein decorin

• Produced by bacteria in a mammal

VlsE• Surface protein that varies antigenically, similar to variable membrane

proteins found in B. hermsii (the causative agent of relapsing fever)

Figure 5: B. burgdorferi

table 1: Features of major surface proteins of B. burgdorferi.

tIcK IntErActIon WItH tHE HoSt

IMMunE SYStEMThe outcome of tick feeding and pathogen inoculation is

governed by the interplay of the tick’s salivary pharmacologic

repertoire and the host’s innate and adaptive immune

responses. Ticks target common pro-inflammatory pathways

of cell activation and recruitment, such as bioactive amines,

chemokines and the complement system. For example,

adult D. reticulates and A. variegatum salivary gland extracts

inhibited human natural killer cells (NK), while I. ricinus had

no effect (37, 38). IL-2, an important growth factor for NK

cells and T cells, is blocked through a putative IL-2 binding

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).

tHE orGAnISMBased on genotyping of isolates from ticks, animals, and

humans, three pathogenic groups of Borrelia burgdorferi

(senso lato) have been identified. In the United States, the

sole cause of human infection is group one, B. burgdorferi

(senso stricto). Group one can also be found in Europe

and Asia. Although all three groups are found in Europe,

the predominant groups are group 2 (B. garinii) and group 3

(B. afzelii). Only groups 2 and 3 have been associated with

Lyme disease in Asia. Although closely related, the disease

patterns observed in humans differ greatly depending upon

the particular Borrelia species involved.

Borrelia species are spirochetes, spiral-shaped organisms

that have a gram-negative cell wall architecture consisting

of two membranes, between which is sandwiched the

peptidoglycan layer (Figure 5) (45). The Borrelia spp. are

longer and more loosely coiled than other spirochetes. Of

the Borrelia spp., B. burgdorferi is the longest (20 to 30 mm)

and narrowest (0.2 to 0.3 mm) and has fewer flagellae (8-

12, 46). The bacteria are actively motile, but the flagellae do

not extend outward from the surface of the bacterium like

those of most gram-negative bacteria. Rather, the flagellae

are embedded into the two ends of the spirochete and are

wrapped around the inside the space between the

cytoplasmic membrane and the outer membrane. Periodic

contractions of these internal flagellae cause the outer

surface to rotate, producing a corkscrew-type motility.

This type of motility may be responsible for the ability of

spirochetes to move out of the skin into the bloodstream and

out of the bloodstream into tissues. Borrelia spp. do not have

LPS in their outer membranes. Instead, the outer membrane

contains a number of lipoproteins, some of which are

designated Osp, for outer surface proteins, A through F. Two

outer surface proteins, Osp A and Osp B, are encoded by the

same 50 kilobase linear plasmid and share 56% sequence

homology (47) (table 1).

tHE Borrelia GEnoMEBorrelia species have linear plasmids as well as the

conventional circular plasmids. Linear plasmids were first

discovered by scientists studying B. burgdorferi, a feature

originally thought to be unique to this genus. In recent years,

however, linear plasmids and chromosomes have been

found in a number of other bacterial groups. The two strands

of the double stranded linear plasmids are covalently closed

at the ends and have sequences similar to telomeres found

on eukaryotic chromosomes. The mechanisms by which the

linear plasmids of spirochetes replicate is still unknown.

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The complete genome of a prototypic B. burgdorferi strain

(B31) was sequenced (48). The total genome is small

(approximately 1.5 megabases) and includes a linear

chromosome of 950 kilobases along with 9 circular and 12

linear plasmids. The plasmid comprises approximately 40%

of the bacterial genome. The organism has a minimal number

of proteins with biosynthetic activity and therefore depends

on the host for much of its nutritional requirements. Analysis

of B. burgdorferi plasmid DNA content reveals that extensive

recombination occurs between plasmids leading to genetic

rearrangements. What role this variability of plasmid-borne

genes plays in adaptation of the bacteria to its various hosts

remains to be determined.

Analysis of the Borrelia genome reveals the absence of any

genes encoding cytochromes or tricarboxylic acid (TCA)

cycle enzymes, which is consistent with the observation that

although B. burgdorferi can grow in the presence of oxygen,

it seems not to need oxygen for growth. Because the bacteria

lack a TCA cycle, they can not make any of the amino

acids whose synthesis requires TCA cycle intermediates.

Given this, it appears odd at first that there were few genes

encoding amino acid transport proteins. This is consistent,

however, with the observation that the bacteria preferentially

take up peptides rather than individual amino acids.

PAtHoGEnESISTo maintain its complex enzootic cycle, B. burgdorferi must

adapt to two markedly different environments, the tick and

the mammalian host. For example, in the mid-gut of the tick,

the spirochete expresses OspA and OspB (48). When the

blood meal is taken, OspC is up-regulated as the organism

migrates to the tick salivary gland and the mammalian host

(49). After injection of the spirochete by the tick and an

incubation period of 3 to 32 days, the spirochete usually

first multiplies locally in the skin at the site of the tick bite.

Several days later, the spirochete begins to spread in the

skin and within days to weeks it may disseminate further.

Bacterial spread within the host is probably facilitated by

the spirochete’s ability to bind human plasminogen and

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).

Initially, the immune response in Lyme disease seems to be

suppressed (57), which may be an important mechanism

in allowing the spirochete to disseminate. Within days to

weeks, peripheral blood mononuclear cells begin to have

heightened responsiveness to B. burgdorferi antigens and

mitogens (58, 59). In vitro, B. burgdorferi is a potent inducer of

pro-inflammatory cytokines, including tumor necrosis factor-

γ and interleukin-1b from peripheral blood mononuclear cells

(60).

The antibody response to B. burgdorferi develops slowly.

The specific IgM response spikes between the third and

the sixth week of infection (61) and often is associated with

polyclonal activation of B cells, including elevated total serum

IgM levels (62), circulating immune complexes (63), and

cryoglobulins (64). Membrane lipoproteins, including OspA,

are mitogenic for B cells (65). The specific IgG response

develops gradually over months to an increasing array of

spirochetal polypeptides (66) and non-protein antigens

(67). Histologically, all affected tissues show an infiltration

of lymphocytes and plasma cells (68). Some degree of

vascular damage, including mild vasculitis or hypervascular

occlusion, may be seen in multiple sites, suggesting that

the spirochete may have been in or around blood vessels.

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.

cLInIcAL SIGnIFIcAncELyme disease occurs in stages, with remissions,

exacerbations and different clinical manifestations at

each stage. Early infection consists of stage 1 (localized

EM) (Figures 1,6) (1), followed by stage 2 (disseminated

infection), and finally stage 3 (late infection or persistent

infection). Stage 3 usually begins months or years after

disease onset, sometimes following long periods of latent

infection (69). In an individual patient, however, the infection

is highly variable, ranging from brief involvement in only one

system, to chronic multi-system involvement of the skin,

nerves, and joints for a period of years.

Figure 6: Erythema migrans rash in Lyme borreliosis

Early Infection: Stage I (Localized

Infection)The first phase, or the skin phase, follows inoculation of the

spirochete into the skin by a tick during its blood meal. The

Ixodes tick that transmits Lyme disease to humans is a pool

feeder in that it does not inject the bacteria directly into the

blood stream but creates a lesion into which blood flows.

(Figure 4E) Thus, the bacteria are originally introduced

into the lower layers of skin. EM, which occurs at the site of

the bite, usually begins as a red macule or papule. In one

study, only 31% of patients (N=314) recalled a tick bite at

the skin site where EM developed 3 to 32 days later (70).

The centers of early lesions sometimes become intensely

erythematous and indurated, vascular, or necrotic. In some

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

been documented that as many as 40% of patients do not

exhibit this characteristic skin manifestation. This initial skin

phase may give the spirochete some protection from the

neutrophils that patrol the blood stream. The ability of the

spirochete to move away from the region may also play a

protective role.

Early Infection: Stage II (disseminated

Infection)The spirochete next invades the bloodstream, leading to

further dissemination throughout the body. Within days

after the onset of the initial EM skin lesion, patients may

experience multiple annular secondary lesions (70, 71).

During this period, some patients develop macular rash,

conjunctivitis, or, rarely, diffuse urticaria. EM and secondary

lesions usually fade within 3 to 4 weeks (range, 1 day to

14 months). EM is often accompanied by malaise, fatigue,

headache, fever, chills, generalized achiness, and regional

lymphadenopathy (70, 71). In addition, patients may have

evidence of meningeal irritation, mild encephalopathy,

migratory musculoskeletal pain, sore throat and cough.

Except for fatigue and lethargy, which are often constant,

the early sign and symptoms are typically intermittent and

changing. After several weeks to months, however, 15%

of untreated patients in the United States develop frank

neurologic abnormalities, including meningitis, encephalitis,

and cranial neuritis, including bilateral facial palsy (72). The

usual pattern consists of fluctuating symptoms of meningitis

with superimposed cranial neuritis, particularly facial palsy.

On examination, such patients usually have neck stiffness.

Facial palsy, or Bell’s palsy, frequently occurs alone and

may be the presenting manifestation of the disease (73).

In Europe, the most common manifestation is Bannwarth’s

syndrome, which includes neuritic pain, lymphocytic

pleocytosis without headache, and sometimes cranial neuritis

(74). Within several weeks after the onset of illness, about

5% of untreated patients develop cardiac involvement (75)

with the most common abnormality as fluctuating degrees of

atrioventricular block (75, 76). However, some patients have

evidence of electrocardiographic changes compatible with

acute myopericarditis (77).

During this stage, musculoskeletal pain is common. The

typical pattern is one of migratory pain in joints, tendons,

bursae, muscle or bones, often without joint swelling.

In addition, a few patients have been described with

osteomyelitis and myositis (78, 79). Conjunctivitis is the

most common eye abnormality in Lyme disease, but deeper

tissues in the eye may be affected as well (70).

Late Infection: Stage III (Persistent

Infection)The third stage of the infection begins when the spirochete

enters tissue throughout the body. At that point, the bacteria

become more difficult to find and may enter a quiescent

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

begin to experience intermittent attacks of joint pain and

swelling, primarily in large joints, especially the knee (80).

Although the spirochete has been cultured from the joint fluid

of only two patients with Lyme arthritis (69), B. burgdorferi

DNA may be detected by polymerase chain reaction (PCR)

in the synovial tissue or joint fluid of most patients (81, 82). B.

burgdorferi–specific T cells, reactive with multiple spirochetal

polypeptides, are concentrated in joint fluid (59, 83). These

T cells secrete primarily the pro-inflammatory Th1 cytokine

IFN-γ. This response is dominant in synovium, a pattern

that leads to a delayed hypersensitivity response (84). From

months to years after disease onset, sometimes following

long periods of latent infection, patients may develop chronic

neurologic manifestations of the disorder (84, 85). The most

common form of chronic central nervous system involvement

is a sub-acute encephalopathy affecting memory, mood, or

sleep (87, 88). Patients with these symptoms often have

CSF abnormalities, including increased CSF protein levels,

evidence of B. burgdorferi antibodies, or both (87, 88).

Borrelia garinii

Borrelia garinii is transmitted by the ticks Ixodes ricinus and

Ixodes persulcatus and appears to be the most neurotropic

of the three Borrelia species. Borrelia garinii may cause

an exceptionally wide range of neurologic abnormalities

including borrelial encephalomyelitis, a multiple sclerosis–like

illness, which is a severe neurologic disorder characterized

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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.

Figure 8: ELISA Assay

by spastic parapareses, ataxia, cognitive impairment,

bladder dysfunction, and cranial neuropathy, accompanied

by antibody production to B. burgdorferi.

Borrelia afzelii

Borrelia afzelii is transmitted by the ticks Ixodes ricinus

and Ixodes persulcatus. In Europe and Asia, B. afzelii may

persist in the skin for decades resulting in acrodermatitis

chronica atrophicans (ACA) (Figure 7) (1). ACA occurs

during the third stage, or late stage, of European Lyme

Borreliosis, and is a skin condition that occurs primarily on

sun-exposed surfaces of distal extremities beginning with

an inflammatory stage with bluish-red discoloration and

cutaneous swelling and concluding several months or years

later with an atrophic phase. Sclerotic skin plaques may

also develop. The frequency of ACA is about 1-10% of all

European patients with Lyme Borreliosis, varying according

to the region of the population sampled. ACA is probably the

most common late and chronic manifestation of borreliosis

in European patients with Lyme disease. Because the

clinical diagnosis of ACA is much more difficult than that of

EM or Borrelia lymphocytoma (BL), the condition is often

underdiagnosed, and, in fact, the ratio of EM cases to ACA

cases may be higher.

dIAGnoSIS

Indirect (Antibody detection tests)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

in the case of Lyme disease. Direct tests are those that

detect the entire bacterium (usually by culture or staining

techniques) or parts of the bacterium such as cell wall

proteins, carbohydrates, intracellular proteins, or nucleic

acids such as DNA or RNA. Both indirect (serology) and

direct tests (PCR) are available for the accurate, sensitive,

and specific laboratory detection of Lyme disease. Because

of the complex nature of the disease, no single test may be

sufficient for all clinical situations.

Medical Diagnostic Laboratories (MDL) offers three serology

tests for the detection of antibodies to Borrelia burgdorferi,

the causative agent of Lyme disease (LD):

1. First step Enzyme-Linked Immunosorbent Assay

(ELISA) screening test

2. Supplemental Western immunoblot (IgG, IgM)

3. C6 Lyme peptide assay (C6LPA)

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 H2SO

4, 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).

The ELISA should only be ordered for patients who have

signs and symptoms consistent with Lyme disease. Equivocal

or positive ELISA tests are recommended by the CDC to

be followed with a supplemental Western blot. However,

some clinicians may wish to use a Western blot test as a

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

antigen. Due to its cross-reactive components, a false

positive reaction will often occur with syphilis patients or other

patients with spirochetal diseases, such as leptospirosis

or periodontal disease, since these treponemes also have

similar gp41 flagellar antigens. This degree of cross-

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.

Figure 9: An IgG positive patient Western blot strip (top) in

comparison to a band locator strip (bottom).

B. Western immunoblot (IgG, IgM) B. burgdorferi

antibodies

Early tests for the laboratory diagnosis of Lyme disease

suffered from a lack of both sensitivity and specificity.

Enzyme linked immunoassays (ELISA) used whole cell

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

1989, the Centers for Disease Control and Prevention (CDC)

recommended that all indeterminate and positive ELISA tests

be confirmed by Western blot (88). Subsequently, tests have

become available which are both more sensitive and specific

but the majority of clinical laboratories still use a whole cell

lysate ELISA screening test with a supplemental Western

blot test performed on indeterminate and positive screening

results.

The Western blot is an antibody test which is capable

of differentiating the antibody response to the variety

of B. burgdorferi antigens. The antigens are separated

electrophoretically and then transferred, or “blotted”, to a

nitrocellulose strip. The strips are exposed to the patient

sample containing antibodies that are both specific to B.

burgdorferi as well as other infectious agents. The presence

of colored bands on the strips is indicative of an antigen/

antibody reaction. The molecular weight of the antigen can be

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

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

test is positive or negative. In the Western blot sample

illustrated in (Figure 9), the top strip is from a patient positive

for IgG and the bottom strip is the band locator strip which

is used for comparative purposes to help properly identify

band location.

The CDC recommends an interpretation of Western

blot based on the work of Dressler et al. (90). The cdc

crItErIA includes:

IgM Significant Bands:

Reactive: At least two of the following three bands must be present: 23, 39, 41.

IgG Significant Bands:

Reactive: At least five of the following bands must be present: 18, 23, 28, 30, 39, 41, 45, 58, 66, 93.

Non-Reactive: Fewer than two bands are present.

* Note that the 31 and 34 kDa B. burgdorferi specific bands represent outer surface proteins A and B, respectively, have been excluded from the IgG criterion. Note also, that there is no indeterminate or equivocal category— five bands are interpreted as positive while four bands constitute a negative result. Included in the IgG interpretive criterion is the 41 kDa band observed in 65% of the normal population.

Tilton et al. (91) and others have proposed the following

ALtErnAtE crItErIA:

IgM significant bands:

Reactive: Two or more of the following four bands must be present: 23, 39, 41, 83/93.

IgG significant bands:

Reactive: Three or more of the following bands must be present: 20, 23, 31, 34, 35, 39, 83/93.

Equivocal: One of the following bands must be present: 23, 31, 34, 37, 39, 41, 83/93

Non-reactive: No Lyme-specific bands present.

www.mdlab.com • 877 269 0090 9

the major differences are:

IgM – This interpretive criterion is similar to the CDC except

that 83/93 has been included as a significant IgM band

that is specific to B. burgdorferi. The Alternate Criteria also

incorporates an “Equivocal” category that includes late

appearing antibodies specific for the 31 kDa OspA and 34

kDa OspB antigens.

IgG – The Alternate Criterion is based on both the number of

bands and the significance of the antibodies detected. For

example, Osp A (31 kDa) and B (34 kDa) are important bands

often seen in late stages of Lyme disease. The alternate

criteria also includes an “Equivocal” category which indicates

that although there are insufficient antibody bands present

for the blot to be reactive, there is significant immunologic

activity observed which may be related to Lyme disease.

To determine the effect of reporting results using the CDC

vs. the alternate criteria (92), commercially available panels

(BBI Diagnostics, CDC) of characterized serum samples

were used. The results indicated that the use of the Alternate

Criteria markedly increased the sensitivity of the Western

blot while only decreasing the specificity slightly.

This raises the question at to whether a Western blot

should be more sensitive or as specific. If the Western blot

is to be used solely for confirmation of ELISA results, then

specificity may be preferred over sensitivity. However, a

specific Western blot with low sensitivity may invalidate a

sensitive and specific ELISA. A Western blot that is both

highly sensitive and specific is desirable for a two-tiered

test scheme. However, despite the recommendations of

the CDC for two-tiered testing, many physicians who treat

patients with Lyme disease do not accept ELISA as a very

sensitive screening test and, therefore, request both ELISA

and Western blot be performed on their patients.

** Please note that BotH the Alternate and the cdc

criteria will appear on all Lyme Western blot result reports

from Medical diagnostic Laboratories. occasionally, this

will cause differences in results; your alternate report

will also include an equivocal category.

c. the c6 Lyme Peptide Antibody test

Commercially available Lyme disease antibody kits, as well

as most research level Lyme disease kits, use a whole cell

lysate as the capture antigen. The capture antigen is used

to coat a solid support such as a microwell plate and binds

specific antibodies that may be present in the serum sample.

These lysates contain multiple epitopes, not all of which

are specific for B. burgdorferi. Because of the immunologic

complexity of a whole cell lysate, a high background is

often present and false positives are therefore a problem.

This is the basis for the lack of specificity inherent to most

enzyme immunoassay (EIA) kits and thereby necessitates

confirmatory immunoblot tests to distinguish Lyme-specific

from non-specific antibodies.

Yet another problem with whole cell ELISAs is the presence

of antibodies to OspA that are vaccine induced. Separating

the vaccine response from the natural host response to

infection becomes very difficult with whole cell ELISAs.

Single or multiple peptide tests use only antigens that may

be specific for B. burgdorferi. These include tests using

recombinant OspA, OspB, and flagellar proteins (91),

recombinant OspC, p39 and flagellar proteins, p83 antigen,

the hook protein of B. burgdorferi (FlgE), p37, and the C6

Lyme peptide recombinant or synthetic antigen (93, 94).

The single peptide Lyme C6 EIA for Lyme disease has

been approved by the Food and Drug Administration (FDA).

The Borrelia burgdorferi C6 peptide antibody test (C6 LPA)

identifies antibodies to a newly discovered, conserved

peptide called C6, which is a component of the variable

surface antigen of B. burgdorferi. The C6 LPA is important

because it detects both IgM and IgG antibodies in patients

with LD but not in vaccinees.

The C6 LPA, like some other peptide assays (the exception

being the OspA EIA), can effectively discriminate vaccinated

from unvaccinated patients with Lyme disease. One

hundred percent of vaccinees tested had a negative Lyme

C6 EIA. The C6 LPA FDA submission reported sensitivities

for acute, convalescent and late stage disease of 74%, 90%,

and 100%, respectively (93, 94). More recent unpublished

data (99) suggest that the sensitivity of the C6 LPA in chronic

Lyme disease may not be as high as originally anticipated

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

become the test of choice for screening purposes. Serum

specimens from patients with early neuroborreliosis are 95%

positive. The early C6 response to infection includes both

IgM and IgG production and may appear soon after a tick

bite (93, 94). Patients with over a dozen different diseases

including systemic lupus erythematosus, non-Lyme arthritis,

syphilis, other spirochetal diseases, and other autoimmune

diseases were uniformly C6 negative. Antibodies produced

by challenge with B. burgdorferi, B. afzelli, and B. garinii

can all be detected with the C6 LPA. Although the latter two

strains are not indigenous to the United States, both are

prevalent in Europe.

All current positive serological screening tests should be

confirmed by Western blot. With more clinical experience,

references:

the high specificity of a positive C6 LPA may eliminate the

need for confirmatory testing (95). The immune response to

the OspA vaccine has been reported to induce antibodies

that bind to multiple proteins of B. burgdorferi (95). Sera

from vaccinated people show multiple low molecular weight

bands in addition to a broad band at 31 kDa on Western

blot. The utility of a Western blot in a vaccinated person is

questionable. Until recently, there has been no “test of cure”

for Lyme disease and the clinical determination of a “cure” is

very controversial. Whether persistent symptoms (persistent

Lyme disease, post-Lyme syndrome, chronic Lyme disease)

after adequate therapy are due to active B. burgdorferi

infection, existing tissue injury, a post-infectious syndrome

or a condition unrelated to Lyme disease is not well defined.

Routine ELISAs do not correlate with the disappearance or

reappearance of symptoms. In contrast, C6 LPA antibodies

diminished by a factor of >4 in successfully treated patients

and <4 in patients who did not respond to appropriate therapy

for Lyme disease. Control tests using a whole cell ELISA or a

p39 recombinant EIA were either unchanged or showed no

significant change in titer as a result of treatment (93, 94). The

results suggest that quantitative C6 LPAs administered over

a period of time may be useful for monitoring a response to

therapy as well as to determine whether the spirochete has

been eliminated. Finally, the question of active vs. inactive

Lyme disease may be resolved because a positive C6 LPA

suggests active infection and a negative test, even with a

positive routine ELISA, suggests inactive infection or no

infection at all, except in very early acute infections (93, 94).

direct tests (Molecular detection via

real-time Pcr)

DNA detection by Real-Time PCR is available from MDL for

whole blood, serum, urine, CSF, and synovial fluid samples.

Four individual assays were developed to differentiate

between the three B. burgdorferi sensu lato genospecies

known to cause Lyme disease in humans, B. burgdorferi

sensu stricto, B. garinii and B. afzelii, as well as the clinically

related, but genomically distinct, B. lonestari.

The ability to distinguish among these pathogens offers a

diagnostic benefit that traditional sero-based testing methods

do not provide. Variations between the genetic composition

of each pathogen make each species distinct from one

another and, as a result, the immune response elicited by

each distinct, too. Antibodies are produced against discrete

regions of proteins termed epitopes that are “presented”

during the immune response. Genetic variations between

pathogenic epitopes allows for the generation of antibodies

specific to a particular pathogen and serves as the basis

of immune memory. This high degree of antibody specificity

serves to limit the diagnostic value of conventional ELISAs.

Real-Time PCR represents a significant advancement

to traditional PCR and has improved the specificity and

sensitivity of diagnostic testing of this type of testing. Like

traditional PCR, real-time assays utilize sequence-specific

primers that allow for the highly specific amplification of DNA

sequences from minute amounts of target material. What

makes this technology superior, however, is the incorporation

of a sequence-specific fluorescent reporter probe that allows

for an accurate quantitation of the reaction following each

amplification cycle.

The limitations associated with conventional Lyme testing are

especially evident when taking into consideration infections

with Borrelia lonestari, a clinically similar, yet genomically

distinct, pathogenic strain. In this instance, serum samples

from individuals determined to be infected with B. lonestari

were unable to recognize B. burgdorferi antigens, rendering

conventional ELISAs useless (96, 97). Despite the fact that

most of these organisms are separated geographically

from one another and, as such, infection with these agents

would be more presumptive, the high rate of travel within

the Unites States and to Europe means that none of these

pathogens should be discounted when a patient presents

with symptoms typical of Lyme disease. The relevance of

such speciation tests is further supported by the report of

an individual from Westchester County, New York who

was infected with B. lonestari during a trip to Maryland and

North Carolina (98). Therefore, in instances where patient

symptoms are suggestive of Lyme disease yet conventional

Lyme tests return negative or ambiguous results, direct

screening via PCR-based technology should be employed.

Borrelia lonestari

Since the mid-1980’s, physicians in non-Lyme disease

endemic areas of the south eastern and south central portion

of the United States have described a Lyme disease-like

illness. Two to twelve days following the bite of the Lonestar

tick, Amblyomma americanum, patients would describe an

erythema migrans (EM)-like rash and mild flu-like symptoms

including fever. This condition is often referred to as southern

tick-associated rash illness (STARI), Master’s disease, or

southern Lyme disease. Based on PCR amplification of the

flagellin and 16s rRNA genes, a new spirochete, Borrelia

lonestari, has been identified. Optimal management of this

condition will depend upon further discoveries regarding its

natural history and etiology.

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A) Life stages of a tick B-c) American dog tick B) Femalec) Nymph d-F) Brown dog tick d) Female E) Larva F) Nymph G-H) Lone star tick G) Female H) Male