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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).
www.mdlab.com • 877 269 0090 3
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.
www.mdlab.com • 877.269.0090 5
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
www.mdlab.com • 877 269 0090 7
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