Borrelia

TAXONOMY Back to top

Borrelia belongs to the order Spirochaetales, which encompasses the

families Spirochaetaceae andLeptospiraceae. Within the Spirochaetaceae, two

genera, Borrelia and Treponema, cause human disease. Borreliae are agents of LBRF and

both tick-borne LB and relapsing fever. The type species of the genusBorrelia is Borrelia

anserina, which causes borreliosis in birds. Based on rrs (16S rRNA gene) sequence

analyses, spirochetes form a distinct entity (division D) within the eubacterial kingdom. They

are neither gram positive nor gram negative. In case of the spirochetes, morphological

criteria and DNA data produce concordant phylogenies, a rare trait in other bacterial groups.

DESCRIPTION OF THE GENUS Back to top

Common Characteristics

Borreliae (Fig. 2 and 3) are similar in length (8 to 30 μm) but wider (0.2 to 0.5 μm) than the

two other human-pathogenic spirochetes, the treponemes and the leptospires (14). They are

highly motile organisms, with corkscrew and oscillating motility enabling movement through

highly viscous mediums such as connective tissue. In contrast to the exoflagella of other

bacteria, the flagella of spirochetes are endoflagella. The endoflagella (7 to 20 per terminus)

are localized beneath the outer membrane and insert subterminally at one end or the other

of the protoplasmic cylinder. The protoplasmic cylinder consists of a peptidoglycan layer and

an inner membrane which encloses the internal components of the cell (14). If cultivable,

borreliae grow slowly under microaerophilic (13) or anaerobic (96) conditions. They

require N-acetylglucosamine and long-chain saturated and unsaturated fatty acids and

produce lactic acid through glucose fermentation (65).

Back to top

Species Diversity

The causative agent of LB, B. burgdorferi, was first described by Burgdorfer et al. in the early

1980s (23). Studies published since 1992 have divided B. burgdorferi sensu lato into 3

prevalent, human-pathogenic species—B. burgdorferi sensu stricto, B. afzelii, and B.

garinii (12)—and 11 other species, some of which have been linked to human cases (Table 1)

(122). In North America, B. burgdorferi sensu stricto is the only human-pathogenic species,

whereas all three species have been isolated from humans in Europe. From central to

eastern Asia, B. garinii and B. afzelii are the agents of almost all human cases of LB.

There is a high prevalence of B. afzelii among human skin isolates from Europe, whereas

isolates from cerebrospinal fluid (CSF) in Europe are most often B. garinii (Table 2)

(39, 104). All three genospecies cause Lyme arthritis in Europe (Table 2) (38, 44, 119).

A few studies have reported the detection of other Borrelia species (B. valaisiana, B.

spielmanii, and B. lusitaniae) in patient samples in Europe (28, 100, 101, 121). Similarly, B.

lonestari, a species carried by a hard tick but genetically more closely related to the

relapsing fever spirochetes vectored by soft ticks, has been reported from the southeastern

United States. This spirochete has been implicated in at least one case of EM, although a

prospective investigation of skin biopsy and serum samples from 30 Missouri patients who

presented with EM-like rashes failed to detect genetic evidence of B. lonestari or B.

burgdorferi (138).

In North America, B. turicatae, B. parkeri, and B. hermsii have been isolated

from Ornithodoros parkeri, Ornithodoros turicatae, and Ornithodoros hermsii ticks,

respectively, but they may be a single species because their DNA-DNA similarity is greater

than 70%. The species status of other cultivable borreliae, such as B. anserina, B.

crocidurae, B. recurrentis, and B. coriaceae, has been supported by greater DNA-DNA

dissimilarity findings (31, 65).

The Genome

The genomes of the borreliae are unusual among prokaryotes in having a small linear

chromosome of approximately 1,000 kb and both linear and circular plasmids. Also atypical

of most bacteria, borreliae have a low G+C content, approximately 30 mol%. The complete

nucleotide sequence of the chromosome and 21 plasmids (9 circular and 12 linear) has been

published for the type strain, B. burgdorferi B31 (43). A total of 59% of the chromosomal

open reading frames (ORFs) have homologs in other bacterial species; in contrast, homologs

have been identified for only one-third of the plasmid ORFs. The genome encodes a basic set

of proteins for DNA replication, transcription, and energy metabolism but, interestingly, lacks

most cellular biosynthetic pathways. Of some surprise is the tremendous number (>150) of

genes that encode putative lipoproteins, suggesting an essential role for these molecules in

the life cycle of the spirochete. Genome analysis of another Lyme disease spirochete, B.

garinii (strain PBi), revealed that most of the chromosome is conserved (>90% DNA and

amino acid level identity) in the two species. Furthermore, two co-linear plasmids (lp54 and

cp26) seem to belong to the basic genome inventory of the Borrelia species that causes

Lyme disease. However, the authors did not find counterparts of the B. burgdorferi plasmids

lp36 and lp38 or their respective gene repertoires in the B. garinii genome (45). The large

linear plasmid lp54 encodes two major outer surface proteins, OspA and OspB, which are

tandemly arrayed in one operon (17). OspC, another major outer surface protein, is encoded

on a circular plasmid (cp26), and sequence analysis of ospC from different strains suggests

that gene exchange might play a role in the diversity and immune evasion of Lyme disease

borreliae (62).

Whole-genome microarray analysis revealed that a total of 215 ORFs, 136 of which are

plasmid borne, were differentially expressed at 23 and 35°C. These findings highlight the

potential importance of plasmid-borne genes in the adaptation of B. burgdorferi sensu lato to

mammal hosts and tick vectors (84). The linear plasmids of B. hermsii and B.

turicatae contain genes encoding outer membrane lipoproteins, called variable major

proteins (Vmp). These genes are silent except when they are translocated to an expression

site immediately adjacent to one of the linear plasmid telomeres. Antigenic variation of Vmplike

proteins due to recombination of vls (Vmp-like small) gene sequence cassettes has also

been described for B. burgdorferi. These vls genes have highly variable regions as well as

highly conserved sequences which encode immunogenic epitopes important for serodiagnosis

(78, 139).

EPIDEMIOLOGY AND TRANSMISSION Back to top

The ecological components that maintain Borrelia species in nature are quite diverse and are

spread throughout the world (Table 1).

Relapsing Fever Borreliae

Most relapsing fever borreliae have rodents as reservoirs and are transmitted by soft-bodied

ticks of the genus Ornithodoros (Fig. 1). One exception, B. recurrentis, the agent of LBRF,

has only humans as reservoirs and is transmitted only by the human-specific body

louse, Pediculus humanus humanus. It has been commonly accepted that B. duttoni, a

prevalent agent of TBRF in east Africa, also had humans as reservoirs, although recent

studies challenge this understanding. Endemic cycles of TBRF between rodents

and Ornithodoros ticks are recognized globally (Table 1). Human infections occur in western

Canada and the United States (reportable in 11 western states), portions of Mexico, Central

and South America, the Mediterranean, Central Asia, and much of Africa. Ornithodoros ticks

are rapid (10 to 30 minutes) and typically nocturnal feeders; human victims most often do

not recall tick bites. Although LBRF had a global distribution only 100 years ago, recent

outbreaks have been limited to parts of east Africa. LBRF is not communicable between its

human hosts, but rapid transfer of the infected louse between persons by direct contact and

shared clothing and bedding enables efficient disease dissemination among crowded

populations, particularly when personal hygiene is compromised.

B. burgdorferi Sensu Lato

The Lyme disease borreliae of B. burgdorferi sensu lato are transmitted by hard-bodied ticks

(genus Ixodes) (Fig. 1). Globally, LB is limited to temperate regions of the northern

hemisphere (Table 1). The prevalence of vector-competent ticks and their infectionpermissive

vertebrate hosts largely defines human risk and case numbers. For example, in

the United States during the 15-year period from 1992 to 2006, 93% of the total cases (n =

248,074) reported to the CDC by health departments were from 10 (Connecticut, Delaware,

Massachusetts, Maryland, Minnesota, New Jersey, New York, Pennsylvania, Rhode Island,

and Wisconsin) of 50 states, and these case incidences were mirrored by B.

burgdorferi sensu stricto infection rates among I. scapularis ticks and reservoir vertebrates

(10).

Ixodes species feed on three different hosts depending on the developmental stage of the

tick. The larvae and nymphs feed primarily on small rodents, whereas adult ticks feed on a

variety of mammals (deer, raccoons, domestic and wild carnivores, larger domestic animals,

and birds). The feeding period of Ixodes ticks is rather long (several days to over a week)

and contributes to their geographic dispersal along with the movement of the host. Birds,

particularly migratory seabirds, can transport the ticks (Ixodes uriae) over very long

distances and thus distribute borreliae (especially B. garinii) worldwide (44).

There appears to be an association between B. afzelii and small rodents and B. garinii and

birds, likely due to different serum sensitivities of the borreliae (59) mediated by

complement regulator-acquiring surface proteins (73). In unfed ticks, B. burgdorferi sensu

lato lives in the midgut. During the blood meal, transcriptional changes are induced in the

borreliae and precede their migration to the salivary glands (108). Migration of spirochetes

from the feeding I. scapularis midgut to the skin of the animal host takes >36 h (32).

For Ixodes ricinus, however, spirochete migration has been observed with ticks feeding for as

few as 17 h (67).

CLINICAL SIGNIFICANCE Back to top

Relapsing Fever

Relapsing fever is an infectious disease with an acute onset of clinical signs and symptoms

including high fever, shaking chills, severe headache, nausea, myalgias, and severe malaise.

Initial physical findings often are conjunctival effusion, petechiae, and diffuse abdominal

tenderness. Fever attacks of 3 to 7 days are interspersed with afebrile periods of days to

weeks. Detailed descriptions and reviews have been published elsewhere for LBRF (98) and

TBRF (8, 37).

LBRF is, in general, more severe than TBRF. An exception to this rule is B. duttoni TBRF in

east Africa. TBRF studies in Tanzania and the Democratic Republic of the Congo have

documented severe morbidity among pregnant women, the young, and the elderly;

pregnancy loss rates of 47% have been reported in some areas of endemicity (30). In TBRF,

up to 13 febrile attacks have been documented, and a rash is more often reported than in

LBRF (28% versus 8%). Splenomegaly, hepatomegaly, and jaundice are observed in 77%,

66%, and 36% of LBRF cases, respectively, whereas these signs are reported in only 41%,

17%, and 7% of TBRF cases. In LBRF, 34% of the patients have respiratory symptoms and

30% have central nervous system (CNS) involvement; in patients with TBRF, these figures

are 16% and 9%, respectively (66). Complications leading to death (mortality rate of up to

40% in LBRF) are acute heart and hepatic failure and cerebral hemorrhage. Disease severity

increases with compromising conditions common to many areas of endemicity.

The initial treatment of relapsing fever cases with appropriate antibiotics may elicit the

Jarisch-Herxheimer reaction (JHR) (37). This reaction, associated with the rapid clearance of

spirochetes from circulation and an overwhelming release of cytokines, typically occurs

within 1 to 4 hours of antibiotic treatment. Signs include hypotension, tachycardia, chills,

rigors, diaphoresis, and sudden elevation of body temperature. Death caused by JHR

associated with LBRF has been reported. While generally not as severe, JHR associated with

TBRF in the United States is reported in approximately 50% of cases. Therefore, patients

with either LBRF or TBRF should be monitored closely upon initial treatment.

Acute respiratory distress syndrome (ARDS) may occur more frequently in TBRF than

previously recognized. In 2004 and 2005, three cases of posttreatment ARDS in patients

with severe TBRF were reported from California, Washington, and Nevada. A retrospective

investigation of 111 TBRF cases reported from these states during the preceding 10 years

revealed two additional ARDS cases, both occurring after 2001. Continued surveillance is

needed to determine whether the risk of ARDS in TBRF is increasing. If so, possible

correlates might include changed medical practices, use of newer antimicrobials, or the

emergence of more-virulent TBRF strains (27).

Lyme Borreliosis

LB can be defined by early localized, early disseminated, and late-stage manifestations

similar to the three stages of syphilis (115). The natural course of untreated B.

burgdorferi infections varies considerably, and clinical manifestations can occur alone or in

various combinations (112, 115). In the majority of cases, the infection is self-limiting, but in

some cases, B. burgdorferi will disseminate to other skin sites, the nervous system, the

joints, the heart, or occasionally, to other organs.

EM at the site of the infectious tick bite is the most common manifestation of early (stage I)

LB and occurs in 60 to 90% of patients. The center of the expanding annular lesion often

fades to produce a bull’s-eye appearance. However, the extension, color intensity, and

duration of EM vary considerably. In Europe, the skin lesion often develops more slowly and

persists longer; hence, the initial description of chronicum migrans (1). One or more general

symptoms, such as fatigue, arthralgia, myalgias, and headache accompany a majority of

primary EM cases (118, 134).

In some patients, hematogenous dissemination of spirochetes to other organs and tissues

occurs within days to weeks of infection (stage II). Patients often feel quite ill and can

present with fatigue, headache, fever, malaise, arthralgia, and myalgia. Multiple (secondary)

erythemata are common in the United States but uncommon in Europe. Neurologic

structures, including the meninges, brain, spinal cord, peripheral nerves, and nerve roots,

are also potential sites of early disseminated infection. In the United States, 15 to 20% of

untreated patients develop neurologic signs, most commonly facial nerve palsy (uni-lateral or

bilateral), meningitis, and radiculoneuropathy. CSF findings in cases of Lyme meningitis

almost always include a mononuclear pleocytosis (10 to 1,000 cells/μl) and elevated protein

concentration. Meningitis, or even facial palsy without meningismus, is more common among

children than adults. Severe encephalitis is occasionally observed in stage II. Bannwarth’s

syndrome is the most common neurologic manifestation of early, disseminated LB in Europe.

The syndrome is characterized initially by intense, migratory or focal, radicular pain,

particularly at night, and by cranial nerve palsy. Paresis of the extremities and the trunk are

less frequent. Further clinical manifestations of stage II may include Lyme carditis, most

often with atrioventricular conduction blocks, and ophthalmic involvement. Borrelial

lymphocytoma, a reddish to livid swelling of the skin that typically occurs in locations such as

the earlobe, nipple, or scrotum, is manifested among some patients in Europe (112, 115).

Lyme arthritis and acrodermatitis chronica atrophicans (ACA), occurring months to years

after the initial infection, are the most common manifestations of late (stage III) disease.

Lyme arthritis can be monoarticular or oligoarticular, typically affecting the knee, and usually

takes an intermittent course. Patients with ACA initially develop an infiltrative stage, followed

by alterations characteristic of the atrophic stage: creased skin with livid discolorations and

plastic protrusion of vessels. ACA is observed almost exclusively in Europe, a finding highly

correlated with B. afzelii infections. Chronic neuroborreliosis is a very rare manifestation of

late (stage III) disease. Paraparesis and tetraparesis are the most common symptoms.

Examination of the CSF reveals a marked elevation of protein concentration with a low to

moderate increase of cells in the CSF. The detection of intrathecally produced specific

antibodies is currently regarded as the best marker of neuroborreliosis (49, 112, 115).

Early manifestations of LB are observed most frequently in the spring, summer, and autumn,

coinciding with tick activity. Late manifestations do not show a seasonal pattern.

COLLECTION, TRANSPORT, AND STORAGE OF

SPECIMENS Back to top

General Remarks for Collection and Transport

For culture, collection and preparation of specimens under sterile conditions are of utmost

importance. Body fluids should be transported without any additives, and biopsy specimens

should be placed in a small quantity of sterile saline or suitable culture medium (see

“Isolation Procedures” below). Samples should reach the laboratory as quickly as possible

(within 2 to 4 h). Before specimens are collected and transported, the laboratory should be

contacted so that details of methodology can be agreed upon. If postal transport is

unavoidable, overnight delivery is recommended. Specimens for laboratory confirmation of

LB are presented inTable 3.

Blood and Serum

For relapsing fever, blood is the specimen of choice. During febrile attacks, borreliae may be

easily detected by dark-field or bright-field microscopy of a wet mount blood sample or a

stained blood smear, respectively (see “Microscopy” below) (Fig. 3). During early febrile

periods, the spirochetemia may reach 106 to 108 cells per ml (65). Blood from acutely ill

patients is also the best source for culture confirmation (31). However, the spirochetemia

diminishes with each successive relapse, and visualization or culture isolation of borreliae is

often unsuccessful during afebrile periods. In contrast to relapsing fever, spirochetemia in LB

patients is below the level of microscopic detection, with estimates of 0.1 spirochetes/ml of

whole blood. The rate of culture recovery from EM patient’s blood has generally been 5% or

less (4). However, in a series of experiments, it was demonstrated that B. burgdorferi culture

recovery from untreated adult patients with EM was better from plasma than from serum or

from an identical volume of whole blood. Approximately 50% of large-volume plasma

cultures from EM patients have yielded B. burgdorferi (135). Serum is suitable for indirect

(antibody) evidence of Borrelia exposure. Specific antibody detection tests are the most

widely utilized tests for laboratory confirmation of LB (see “Serologic Tests” below).

Serodiagnosis of relapsing fever is performed only in a few specialized laboratories.

Cerebrospinal Fluid

Patients with suspected Lyme neuroborreliosis (LNB) may have evidence of immunoglobulin

synthesis againstB. burgdorferi antigens in the CSF, elevated CSF inflammatory cells (usually

lymphocytes, monocytes, or plasma cells), and elevated protein. CSF along with serum

drawn at the same time should be obtained for laboratory demonstration of Borrelia-specific,

intrathecal (CSF/serum antibody index) antibody production (see “Serologic Tests” below).

For culture or PCR, detection rates are only about 20% (76). Positive PCR results with CSF

also seem to correlate inversely with the duration of neurologic disease. Among

neuroborreliosis patients, 7 of 14 (50%) with a disease duration of less than 2 weeks had a

positive PCR result compared with only 2 of 16 (13%) patients in whom the illness duration

was greater than 2 weeks (P= 0.045) (76).

Synovial Fluid or Synovial Biopsy Specimens

Investigation of synovial fluid or a synovial biopsy specimen by PCR can be useful in special

circumstances where Lyme arthritis is suspected or the efficacy of antibiotic treatment is

questioned (see “Nucleic Acid Detection Techniques” below) (4, 82). Culture is usually

negative, with few exceptions. Due to the high protein permeability of the synovium,

synovial fluid and serum display roughly equivalent antibody titers. Thus, it is sufficient to

monitor antibody in serum.

Skin Biopsy Specimens

Skin biopsy samples are the best sources for isolation of B. burgdorferi; spirochetes can be

isolated in most untreated cases of EM and acrodermatitis (Table 4). In cases of EM, culture

success is highest (up to 86%) with biopsy samples taken close to (4 mm inside) the

expanding border of the lesion (16), although this is primarily a technique for research, not

for routine diagnosis. There are indications that the number of spirochetes in the skin is

rather low or unevenly distributed, since an increase in the sensitivity is observed if more

than one biopsy sample is investigated (140). Without treatment, B. burgdorferi sensu lato

can persist for long periods in the skin, as shown by isolation from a 10-year-old

acrodermatitis lesion (7). Biopsy samples (taken after thorough disinfection of the skin)

should be sent in a small amount of sterile saline or Barbour-Stoenner-Kelly (BSK) medium

(with or without rifampin) as soon as possible to a microbiology laboratory capable of

culturing B. burgdorferi.

Other Materials

Ticks are often tested for borreliae as part of epidemiological studies to assess risk to human

populations in a given geographic area. Although specialized laboratories offer diagnostic

services for individual ticks, detection of spirochetes within ticks by PCR or other methods

has not been shown to provide clinically useful information.

DIRECT EXAMINATION Back to top

Microscopy

Direct microscopic visualization of borreliae in clinical samples is applicable only to cases of

relapsing fever. During acute phases, spirochetemia often reaches 106 to 108 borreliae/ml,

and motile spirochetes can be visualized by dark-field microscopy from wet preparations

made from a drop of blood. This simple confirmatory test is often overlooked because of the

increasingly common use of automated differential blood counts. Spirochetes can be

visualized by stained (e.g., Giemsa) thin or thick films (Fig. 3). Detection of low-level

spirochetemias may be assisted by a microhematocrit concentration technique. The

hematocrit capillary is filled 75% with anticoagulated (e.g., EDTA or citrate treated) blood

and centrifuged for 2 min. The buffy coat is then examined directly under the microscope at

×400 to ×1,000 (132). Failure to observe spirochetes does not rule out disease, and culture

isolation (see “Isolation Procedures” below) can be considered.

Antigen Detection

Enzyme-linked immunosorbent assay and immunoblotting have been used for the detection

of borrelial antigen in body fluids, including CSF and urine (29, 60). However, a commercial

assay for antigen in urine was shown to lack reproducibility, and its use is not recommended

(72).

Nucleic Acid Detection Techniques

Nucleic acid amplification techniques (NAAT) may serve as an adjunct to clinical diagnosis

but should be restricted to experienced and specialized laboratories (102, 133). A variety of

chromosomal and plasmid targets for NAAT have been developed (for reviews, see

references 4, 35, 47, and 105). For PCR, an analytical sensitivity of approximately 10 to 20

borreliae per test sample has been demonstrated. Test sensitivities for both NAAT and

culture are greater with tissue specimens than with body fluids, except for synovial fluid,

with which NAAT is superior. Sensitivities of 96% (82) and 86% (19) were reported for NAAT

with synovial fluid from American patients with Lyme arthritis. European authors found NAAT

sensitivities ranging between 50 and 70% (38, 44, 97, 119). Patients with Lyme arthritis are

nearly always seropositive, so PCR of synovial samples is not used as a primary diagnostic

technique. A positive PCR result after antibiotic therapy is of uncertain significance, since the

presence of B. burgdorferi DNA does not necessarily mean that spirochetes are viable (19).

With skin biopsy and CSF specimens, NAAT demonstrated diagnostic sensitivities of

approximately 60% and 20%, respectively (20, 39, 76). A prospective study of PCR and

culture detection of B. burgdorferi in EM biopsy samples from Slovenian patients showed

comparable sensitivities (36% culture positive in modified Kelly Preac-Mursic medium, 24%

culture positive in BSK II medium, and 25% PCR positive) (91). PCR targeting ospA,a

plasmid-borne gene, is more sensitive than flagellin PCR, which uses a chromosomal target

(88, 140). Borreliae can shed blebs containing plasmids, leading to greater abundance of

plasmid than chromosomal genes.

PCR amplification of B. burgdorferi sequences from urine has been described (99, 105) but is

not recommended. Although Borrelia-specific DNA was demonstrated in over 70% of skin

biopsy samples from patients with florid EM, parallel testing of urine samples was uniformly

negative (20).

ISOLATION PROCEDURES Back to top

Many Lyme and relapsing fever borreliae are successfully cultured in artificial media.

However, for diagnostic purposes, culturing is a slow, time-consuming method characterized

by low sensitivity, especially from body fluids of patients with LB (Table 4). For these

reasons, culture attempts are most often limited to research applications and performed by

reference laboratories (e.g., the National Reference Center for Borreliae in Germany and the

Centers for Disease Control and Prevention [CDC] in the United States).

Several media (modified Kelly medium, e.g., BSK II, BSK-H, or modified Kelly Preac-Mursic)

(13, 92, 96) are capable of supporting growth of borreliae. It is important to verify the

quality of each lot of medium by growing a reference strain from a small inoculum (<10

cells). Optimum growth (the generation time of B. burgdorferi is about 7 to 20 h) in these

media is obtained at 30 to 33°C under microaerophilic conditions. Positive cultures from EM

and synovial biopsy or fluid samples (blood and CSF) may be obtained in as few as 4 days,

but most isolates require several weeks of incubation and negative cultures should be

monitored by dark-field microscopy (Fig. 3) for at least 6 weeks.

IDENTIFICATION Back to top

Molecular Techniques

Relapsing fever borreliae have been typed on the basis of DNA-DNA reassociation analysis

and flagellin gene analysis (31, 65). B. burgdorferi has an arrangement of its rRNA genes (a

single rrs and tandemly repeated rrland rrf genes) which distinguishes it from the relapsing

fever borreliae (which have single copies of each) (109). Sequencing of 5S-23S intergenic

spacers and a number of genes, pulsed-field gel electrophoresis of large restriction

fragments, PCR, and restriction fragment length polymorphism analysis of multiple targets

have all been utilized for species differentiation (15, 39, 77, 93, 122, 129, 130). However, in

most cases, diagnosis and effective management of individual patients are independent of

species determinations beyond the Lyme disease and relapsing fever groupings.

Immunological Techniques

Serotyping methods to identify Borrelia species and strains within a species have been

described (125, 126). However, as with molecular techniques, diagnosis and effective

management of individual patients have yet to utilize characterizations beyond the Lyme

disease and relapsing fever groupings.

SEROLOGIC TESTS Back to top

Borrelia Antigens and Human Humoral Immune Response

B. burgdorferi possesses at least 30 immunogenic proteins which include the outer surface

proteins A to F, a number of tissue binding proteins, and components of the flagellar

apparatus. Proper detection and interpretation of the humoral response against B.

burgdorferi must consider several variables. All LB genospecies (or strains within a

genospecies) do not produce qualitatively or quantitatively identical sets of antigens. In

serologic assays that utilize whole-cell culture extracts as the source of reactive antigens,

these variables may result in different sizes of a given antigen (e.g., OspC, 21 to 25 kDa),

quantitative antigen differences, or even their absence. This is particularly problematic in

Europe and Asia, where multiple genospecies are present. Thus, it is imperative, even in

North America, that diagnostic laboratories and manufacturers of serologic assays verify that

all diagnostic antigens are present in relevant amounts. In the case of Western immunoblots,

diagnostic antigens must also be discernable from each other. For many diagnostic proteins,

sequence heterogeneity, even between strains in a given genospecies, may result in amino

acid variations and reduced detection of an antibody response from a patient with a

heterologous infection. Again, OspC serves as an example: with 21 major OspC types

recognized among North American and European isolates (111), patient reactivity in an

assay with one selected OspC type may not be sufficiently cross-reactive to enable its

detection (61, 137). In addition to genospecies and strain-dependent protein profiles as well

as antigenic heterogeneity, many antigens are variably expressed in response to

environmental cues both in culture and during infection. OspC and VlsE serve as examples;

while both of these potent immunogens are expressed during early infection, they are

variably produced in culture, often in very low amounts. Thus, their presence in diagnostic

assays must be verified. Similarly, while OspA expression is turned off in early infection (55)

and is therefore an insensitive marker of this stage of disease, it is an abundant protein in

most cultures. During progression to later stage II and III disease, particularly in North

America, expression of OspA is often triggered and patient antibody to this antigen is

strongly correlated with arthritic involvement (70).

Among B. burgdorferi immunogenic proteins, some have both heterogeneous and conserved

antigen epitopes. Despite the overall antigenic heterogeneity of OspC, its C-terminal 10

amino acids harbor a highly conserved and immunodominant epitope (pepC10) which has

been used successfully in peptide based serodiagnostic enzyme immunoassays (EIAs) (81).

Finally, at least one protein of great and recent diagnostic interest is capable of switching

antigen epitopes during infection. The VlsE (variable major-protein-like sequence expressed)

of B. burgdorferi is a surface lipoprotein that is expressed early in infection. It contains both

variable and invariable regions, and extensive antigenic switching within the variable regions

likely contributes to immune evasion (11, 83, 139). Nonetheless, and of some surprise,

studies in the late 1990s found that LB patients developed strong antibody responses to

VlsE, particularly to the sixth invariant region of the protein (78), and that this region was

highly conserved among the three major LB genospecies. These findings served as the basis

of EIAs in which synthetic peptides representing the sixth invariant (or conserved) region,

C6, were developed. Accumulating published studies over the last 10 years have shown that

VlsE and C6-based assays have high sensitivities in most stages of LB and suggest that they

may serve as future, single-tier assays for serodiagnosis (4, 9, 46, 78, 106, 117).

The earliest immunoglobulin M (IgM) responses to all B. burgdorferi infections are directed

against OspC (21 to 25 kDa), the flagellar antigens, p41 (FlaB) and p37 (FlaA), and p35

(BBK32, fibronectin binding protein) and are typically detectable within the first few weeks.

Detectable IgM against BmpA (39 kDa) is in part strain dependent and most often appears

after the response to OspC, FlaB, and FlaA (2, 4, 34, 40). Although the level of IgM antibody

to most spirochetal antigens peaks within the first weeks, it often persists at detectable

levels for many months.

The IgG response increases and broadens slowly over the first weeks of disease. Among the

reactive antigens to which there is an early IgG response are OspC, p35 (BBK32), p37

(FlaA), VlsE, and p41 (FlaB) (2,5, 74, 78, 86). During early disseminated (stage II) disease,

IgG levels increase, and reactivity against Osp17 (DbpA, decorin binding protein A), p39

(BmpA), and p58 often appears (53). The late-stage immune response (stage III) is

characterized by IgG antibodies to a wide variety of antigens (34, 53). Approximately 80% of

the sera from European patients with late disease (arthritis and ACA) react with p14, Osp17

(DbpA), p21 (not OspC), p30 (not OspA), p39, p43, p58, and p83/100 (homolog of p93)

of B. afzelii strain PKo (53). Similarly, among North American patients with chronic

neurologic abnormalities or arthritis, close to 100% react with 5 or more of the diagnostic

antigens p18, p23 (OspC), p28, p30, p39 (BmpA), p41 (FlaB), p45, p58, p66, and p93

(4, 34, 117).

Notable differences in late-stage disease antibody responses between European and North

American patients include those to OspC and OspA. While IgG antibodies against OspC are

detected in only 20% of European patients with late-stage disease (53), the frequency of IgG

reactivity to OspC in American patients with late-stage disease is 48% (34). Similarly, while

only 5 to 7% of European patients with late-stage disease are reactive to OspA (53, 127)

over 40% of American patients with late-stage disease are reactive to this antigen (5, 34).

Two-Step Approach in Serodiagnosis

For serodiagnosis of LB, a two-step approach is recommended by the Association of State

and Territorial Public Health Laboratory Directors (ASTPHLD) and the CDC (25, 63). All

serum specimens submitted for Lyme disease testing should be evaluated in a two-step

process, in which the first step is a sensitive serologic test, such as an EIA or

immunofluorescence assay (IFA). Specimens found to be negative should not be tested

further. All specimens found to be positive or equivocal by a sensitive first-tier test should be

further tested by a standardized immunoblot procedure (25). This procedure is also

recommended in the MiQ LB standard published by the German expert group on the

diagnosis of LB of the German Society for Hygiene and Microbiology (DGHM) (Fig. 4) (133).

The concept of a two-step approach, which aims at increasing the predictive value of a

positive result with each step, requires that the tests be performed in succession (64,133).

Omitting the first step, a quantitative assay, and proceeding directly with qualitative

immunoblots reduces the specificity of the procedure.

Immunofluorescence Assay

For the IFA, borreliae fixed on glass slides are used as the antigen. IFA for serodiagnosis of

relapsing fever, however, is challenging, since expression of the major membrane proteins is

variable. The specificity of IFA serodiagnosis for Lyme disease may be improved by

adsorption of sera with Treponema phagedenis sonicate (IFA-ABS) (133). For the IgM test,

pretreatment of the sera with anti-IgG immune serum is recommended to avoid falsepositive

test results due to rheumatoid factor as well as false-negative results due to high

IgG antibody levels. As in all antibody detection assays, it is important to verify expression of

OspC within the antigen source cultures. Although IFA is relatively easy to perform, it is not

easy to standardize, and evaluation of test results requires expertise not always available in

the routine laboratory. In general, antibody titers of ≥64 and ≥256 are regarded as positive

on the IFA-ABS and unadsorbed IFA, respectively. Sera from patients with syphilis are often

positive in the unadsorbed assay and are rarely positive on the IFA-ABS (133).

Enzyme Immunoassay

Different modifications of the EIA have been used for the diagnosis of LB. In the indirect EIA,

antigen is used to coat the plates, followed by incubation with patient serum, enzymelabeled

anti-IgM or anti-IgG, and the EIA substrate. Capture IgM-EIA (μ-capture EIA) has

been specially designed to avoid false-positive reactivity due to rheumatoid factor (52).

Rheumatoid factor false-positive reactivity can also be overcome by pretreatment of the sera

with anti-IgG (127). EIA has the advantage of objective measurement, quantification, and

high throughput. Many different antigen preparations have been used, including whole-cell

sonicates (103), isolated flagella (50), detergent extracts (127), recombinant protein

antigens (68, 75, 127), and synthetic peptides (78). Use of crude antigen preparations, such

as whole-cell sonicates, often results in unacceptable specificity. Improved tests which utilize

enriched, specific, or recombinant protein antigens are now widely used. Tests using an octyl

β-D-glucopyranoside detergent extract and Reiter treponeme absorbent, isolated flagella,

recombinant VlsE, or the C6 peptide of VlsE are commercially available (Dade-Behring,

Marburg, Germany; Dakopatts, Copenhagen, Denmark; Diasorin, Turin, Italy; and

Immunetics, Boston, MA). Since VlsE is not present in relevant amounts in cultivated

borreliae, recombinant VlsE has been added to whole-cell extracts to increase sensitivity in

some products (Dade-Behring).

Immunoblotting

The Western immunoblot is regarded as a supplementary (United States) or confirmatory

(Europe) assay. This implies that it should be employed only when a screening assay is

reactive (positive or indeterminate, sometimes called equivocal). Western immunoblotting

enables assessment of the humoral immune response to protein antigens as separated by

sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antigen preparations

for Western immunoblotting include whole-cell lysates or recombinant protein antigen

mixtures that are resolved (largely by molecular weight) by SDS-PAGE and then transferred

to blot membranes. Patient antibody against dozens of borrelial antigens can be discerned by

the experienced diagnostic laboratorian. However, the procedure is considered technically

complex. An alternative test format is the line immunoblot, whereby recombinant or native

borrelia antigens, which have been resolved or purified by means other than SDS-PAGE, are

directly striped on membranes for immunoprobing. This approach enables discrete spacing or

placement of individual antigens in quantified deliveries on the membrane and avoids the

overlap of comigrating antigens that often complicate reading on Western immunoblots. With

either method, Western immunoblot or line immunoblot, it is imperative that antigen

identification and cutoff (minimal band intensity) controls are employed in each diagnostic

run. These calibration controls may include antibody preparations provided with commercial

kits, monoclonal antibodies (MAbs) available from commercial and other sources (e.g., CDC),

or calibrated patient samples.

Numerous immunoblot tests which use antigens of various strains or genospecies of B.

burgdorferi sensu lato are commercially available. The ASTPHLD and the CDC, as well as the

DGHM, have published recommendations for interpretation of the Borrelia immunoblot

(25, 133). In the United States, immunoblot interpretation rules have been recommended

which refer to detection of antibody against whole-cell antigens of specific B.

burgdorferi sensu stricto strains (34, 40). The IgM immunoblot is interpreted as positive if

≥2 bands of the following proteins are reactive: p23 (OspC), p39 (BmpA), and p41 (FlaB).

The IgG blot is interpreted as positive if ≥5 bands of the following proteins are reactive: p18,

p23 (OspC), p28, p30, p39 (BmpA), p41 (FlaB), p45, p58, p66, and p93. If the immunoblot

is used within the first 4 weeks of disease onset (early, stage I or II), both IgM and IgG

immunoblots should be performed. Due to specificity concerns of the IgM immunoblotting

criteria potentially yielding false-positive findings in persons with a low pretest likelihood of

infection, initial recommendations limited application of the IgM Western blot to the first 4

weeks of infection. Beyond this time point, a more-specific IgG-reactive Western blot is

expected. Recent studies, however, indicate that some patients do not develop a robust IgG

response during the first 4 weeks of infection (117).

Interpretation of the antibody response among European patients is complicated by the risk

of infection with different Borrelia species. In addition, immunoblot studies have shown that

the immune response to European infections, compared with North American infections, is

restricted to a narrower spectrum of Borrelia proteins (33). Interpretive rules defined in a

species- and strain-specific manner have been determined (53, 62) and independently

corroborated (59). B. afzelii strain PKo is preferred to PBi (B. garinii) and PKa2 (B.

burgdorferisensu stricto) strains because it permits a two-band criterion for the IgG test: at

least two bands positive for p14, p17 (DbpA), p21, OspC, p30, p39 (BmpA), p43, p58, and

p83/100 (Fig. 5) (133). According to the general Deutsches Institut fur Normung (DIN)

recommendations on the immunoblot (DIN 58967, part 40), at least a two-band criterion

should be required for the positive interpretation of the IgG immunoblot. In IgM

immunoblots, a detectable immune response is restricted to only a few bands. Therefore, the

IgM blot is regarded as positive if there is strong reactivity to OspC (133). Specific DIN

recommendations for the Borreliaimmunoblot (DIN 58969, part 44) have been published

which include new antigens (i.e., VlsE) and the line immunoblot as a new technique.

Detection of Intrathecally Produced (CSF) Antibodies

Approximately 15% of untreated LB patients will develop neurologic manifestations. LNB has

been divided into early disseminated and late stages. Both the CNS and peripheral nervous

system, as well as blood vessels and meningeal coverings, may be involved in either stage.

Laboratory testing should only be used to confirm the diagnosis, and the presence of B.

burgdorferi-specific antibody in CSF or serum may be indicative of past or present infection.

The pattern of nervous system involvement is largely stage dependent and may affect the

correlation between clinical spectrum and serologic test utility. Thus, careful evaluation of a

thorough clinical history and presentation are critical to the selection of appropriate

diagnostic tests and proper interpretation of their findings (49, 54, 85, 94).

In patients in whom the CNS is involved, there should be evidence of CNS inflammation.

Rarely, this may be localized to the brain or spinal cord, but in most cases, it involves the

CSF and is evidenced by pleocytosis, elevated protein concentrations, and in cases of

protracted infection, anti-borrelia-specific immunoglobulin synthesis. In contrast, for

peripheral nervous system-limited disease, the CSF findings may be normal, as it is in most

patients who have toxic-metabolic encephalopathy (49).

Although <10% of LNB cases are culture confirmed, subtle differences in clinical presentation

between European and North American LNB are linked to causative genospecies. North

American cases are limited to B. burgdorferi sensu stricto, while most European culturepositive

LNB cases are B. afzelii and much smaller percentages of cases are B. garinii and B.

burgdorferi sensu stricto.

The triad of early disseminated LNB, also known as meningoradiculoneuropathy, includes

aseptic meningitis, cranial neuropathy, and radiculoneuritis; these may occur singularly or in

combination. The single most common presentation of early disseminated LNB in North

America is meningitis. Examination of the CSF shows mononuclear pleocytosis and elevated

protein. CSF-specific anti-B. burgdorferi (IgA or IgG) immunoglobulin is demonstrated in 80

to 90% of patients. Standard two-tier serology in these patients is also most often positive.

Cranial neuropathy occurs both in North America and Europe in about 10% of early

disseminated LNB cases. Most frequently, this involves the facial nerve and is manifested by

unilateral or bilateral facial palsy. Only about 50% of these cases will demonstrate CSF

pleocytosis. European early disseminated LNB often presents as Bannwarth’s syndrome and

is highly associated with B. garinii infection. This radiculoneuropathy also occurs in up to 5%

of untreated North American LB patients. Most patients with Lyme radiculoneuritis are

reactive in two-tier serologic testing, and CSF findings include pleocytosis and B. burgdorferispecific

antibody (49, 54, 85, 94, 113).

Late neurological manifestations usually develop months to years after initial infection.

Encephalopathy is more common in North America, while encephalomyelitis is more frequent

in Europe. For cases of late encephalopathy, serum immunoreactivity is nearly universal,

while CSF pleocytosis, elevated protein, and B. burgdorferi-specific antibody are found in

only 5%, 20 to 45%, and ~50% of cases, respectively. Most cases of chronic

encephalomyelitis are reported from Europe, although North American cases have been

described. In these cases, CSF pleocytosis and marked B. burgdorferi-specific antibody are

almost universal.

Although there are no well-accepted criteria for seroconfirmation of neuroborreliosis in the

United States, detection of intrathecal Borrelia-specific immune response is a valuable tool

and is widely utilized in Europe (18). Methods taking into account potential dysfunction of the

blood-CSF barrier, a common finding in neuroborreliosis, are required for accurate

assessment of intrathecal antibody production. Long-used procedures for detection of specific

intrathecal antibody production in the diagnosis of neurosyphilis have been modified for the

diagnosis of neuroborreliosis (51, 114, 131). The most frequently used method is the

determination of the CSF/serum antibody index (specific antibody index [AI]). CSF and

serum must be obtained at the same time. By calculating the AI, CSF and serum are

compared with regard to the portion of pathogen-specific IgG antibodies in the total IgG

content. An AI of ≥2.0 is considered significantly elevated (79, 133). Lower indices (e.g.,

≥1.3) are also considered significant by some investigators. False-positive AI results are

likely with neurosyphilis patients when tested with whole-cell or flagellum sonicates in EIA.

Here, EIAs with T. phagedenis adsorption (Dade-Behring) or recombinant antigens not crossreacting

with Treponema pallidum can be helpful for differential diagnosis. Other suitable

methods for determination of intrathecal antibody production are the μ- or γ-capture EIA

(Dakopatts) (51) and the IgG-matched immunoblot (131). The latter allows comparison of

the antibody spectrum (against various Borrelia proteins) in serum and in CSF and thus

permits conclusions as to the specificity of the intrathecal antibody response.

Vaccination: Past, Future, and Impact on Serology

The recombinant OspA vaccine (LYMErix), was withdrawn from the United States market in

2002. However, persistent titers among previous vaccinees may still be encountered and

complicate interpretation of whole- cell-based serologic tests (3). Use of recently available

commercial recombinant immunoblots will avoid false test results among OspA vaccinees.

Controversial Methods

A variety of diagnostic approaches have been developed as alternatives or adjuncts to the

more widely practiced methods described above. T-lymphocyte proliferation assays have

been used in various scientific studies to investigate the human T-cell response

to Borrelia antigens (71). However, T-lymphocyte proliferation assays cannot be

recommended as diagnostic tests due to their cumbersome nature and concerns about their

specificity and standardization (26, 57, 124, 141). Antigen detection tests also are not

recommended, as discussed above.

Detection of B. burgdorferi-specific antibodies in immune complexes has been proposed to be

superior for serodiagnosis of acute Lyme disease (107) and as a marker of active infection

(22). Recent work demonstrates that test results for antibodies precipitated from serum as

immune complexes are highly correlated with enzyme-linked immunosorbent assay results

obtained using unprocessed serum and are not more likely to reflect active infection than

standard serology (80). Transformation of B. burgdorferi into spheroplasts (L-forms) in vitro

in response to deprivation of serum or culture in CSF has been observed (6,21). When

visualized under a microscope, spheroplasts sometimes appear to be enclosed in a sac, so

they have also been called “cysts.” If apparently pure L-forms are injected into mice, they

are infectious (48). The clinical and diagnostic significance of L-forms has not been

demonstrated but warrants further study. There are commercial tests offered that purport to

specifically detect cell wall-deficient or “ cystic” forms of B. burgdorferi by IFA (123a) and

culture (90), but they have not been validated with appropriate controls and are not

recommended.

ANTIMICROBIAL SUSCEPTIBILITIES Back to top

The antimicrobial susceptibility of Borrelia species has been studied intensively in vitro

(65, 95). Standard methods for the determination of the minimal bactericidal concentration

have not been established. However, there is general agreement on the in vitro susceptibility

of borreliae to antimicrobials, as follows. B. burgdorferisensu lato is susceptible to

macrolides, tetracyclines, semisynthetic penicillins, and the expanded- and broad-spectrum

cephalosporins; moderately susceptible to penicillin G and chloramphenicol; and resistant to

trimethoprim, sulfamethoxazole, rifampin, the aminoglycosides, and the quinolones (65). No

significant differences between the Lyme disease borreliae and relapsing fever borreliae (B.

hermsii and B. turicatae) were found with regard to penicillin G, amoxicillin, ceftriaxone,

erythromycin, azithromycin, doxycycline, or tetracycline (65). There is no indication for

routine antimicrobial susceptibility testing in either Lyme disease or relapsing fever.

Recommendations for Antibiotic Therapy

All clinical manifestations of B. burgdorferi infection should be treated with antibiotics. The

antibiotic, dosage, duration, and route of application depend on the clinical picture and stage

of the disease (123, 136). In cases of solitary EM, oral treatment with doxycycline,

amoxicillin, or cefuroxime axetil is recommended. In acrodermatitis, the same antibiotics and

daily doses as in EM are recommended. In arthritis, oral treatment with doxycycline may be

tried first, but in cases of poor therapeutic response, patients should be treated

intravenously with cephalosporins or penicillin G. Intravenous cephalosporins or penicillin G

is also recommended for stage III neuroborreliosis.

EVALUATION, INTERPRETATION, AND REPORTING OF

RESULTS Back to top

General Aspects

Clinical criteria (case history and clinical findings) are decisive factors in the diagnosis and

ordering of microbiological laboratory testing. The predictive value of laboratory tests is

directly related to the pretest probability. It should be kept in mind that the lower the

probability based on the clinical diagnosis, the lower the predictive value of a positive test

result. For example, a negative serologic result has a high negative predictive value for Lyme

arthritis, since nearly all cases are seropositive. Whether or not a positive test corresponds

with the patient’s presentation is a question that can only be answered by the clinician, e.g.,

by means of clinical case definitions applied to the various manifestations of LB. Therefore,

the laboratory report should not contain any therapy recommendations.

Serologic Report

The serologic report should contain the following points:

1. Recording of individual test results. Results of the first assay, generally an EIA, are

reported as positive, indeterminate, or negative. The immunoblot results are reported as

positive or negative. In the case of a positive result, the reactive diagnostic bands may be

reported (135). Caution against overinterpretation of minimally reactive blots must be

emphasized (e.g., IgG reactivity against p41 is expected in approximately 50% of healthy

adults in the United States and Europe and is excluded from the European scoring criteria).

2. Assessment of the final result of the two-step approach regarding its immunodiagnostic

significance (e.g., whether specific antibodies have been detected or not).

3. Assessment of serologic findings as to the stage of the immune response, as far as test

results allow pertinent statements to this effect (see below).

4. Recommendations for further reasonable diagnostic methods (PCR or culture) or for

serologic follow-up, if indicated.

Patterns of Serologic Results in Various Stages of LB

Antibody tests performed in the early stage of LB, particularly in cases lacking evidence of

spirochetal dissemination, may show a negative or an indeterminate result (Table 5), often

due to insufficient time for the full evolution of the immune response. In some cases,

seroconversion does not occur until after initiation of treatment. Serologic testing of patients

with EM alone is not recommended because of the low predictive value of a negative result

and the highly characteristic appearance of most rashes. In the presence of a suggestive

clinical presentation and inadequate response to therapy, serologic testing is warranted up to

6 weeks after onset of disease. During the first 4 weeks of a positive clinical correlation,

detection of IgM is consistent with an active infection. A robust IgG response is expected

thereafter. Usually, only a few bands (IgM and/or IgG) are detected by immunoblotting

during the first weeks of early disease. In late disease, a positive test for IgG antibodies is

mandatory for seroconfirmation and the IgM test is not useful for establishing the diagnosis;

the absence of IgG rules out the diagnosis of late Lyme disease even in the presence of IgM.

False-positive IgM results due to a polyclonal B-cell activation immune response in the

context of herpesvirus infections or autoimmune diseases and rheumatoid disorders also

need to be considered. In many cases, the origin of such IgM responses remains unclear.

Both IgM and IgG may persist for many months, and their presence may be compatible with

past, asymptomatic, spontaneously resolved, or treated and clinically cured infections. Such

patterns are often found among members of high-risk groups with frequent tick exposure

(for example, forest workers) who do not show any clinical manifestations.

Influence of Antimicrobial Therapy on Serodiagnosis

Clinicians are often tempted to order repeated posttreatment serologies in an effort to

correlate cure and decreasing antibody titers. However, IgG antibodies against B.

burgdorferi (especially those against whole-cell antigens) persist for a long time even after

successful therapy. Significant titer changes can only be expected several months after the

end of therapy; in cases of late manifestations, even years may elapse. Moreover, a

decrease in antibody titer does not rule out persistence of the pathogen. Since there is

practically no indication for follow-up serologic tests, therapeutic success should be based on

clinical criteria. A fourfold decline in titer of antibody to VlsE peptide C6 was shown to be an

indicator of successful therapy for early Lyme disease (89), but this was not demonstrated

for late disease (87) or post-treatment Lyme disease syndrome (42).

Sources of Error in Serodiagnosis

False results, both negative and positive, can occur from the test itself or the nature of the

immune response. Seronegative results within the first days of disease are the norm. In

Europe, differences between the test antigen and the species causing infection can also

contribute to seronegative findings. Deficiencies in diagnostic antigen expression among

cultivated borreliae will compromise the sensitivity of tests (important diagnostic antigens

such as OspC and DbpA are often not expressed in cultivated borreliae, and VlsE is poorly

expressed in vitro). The high background reactivity of many first-generation whole-cell-based

assays often results in lower specificity and, therefore, frequent false-positive results. Crossreactivity

with treponemes can be largely avoided by use of Reiter treponeme adsorbent,

although syphilis serology should be performed in cases where treponeme exposure cannot

be ruled out. Second- and third-generation assays with improved sensitivity and specificity

are preferable to the first-generation tests. Nonetheless, critical assessment of pretest risk

factors, clinical history, and presentation will provide the best guidance for laboratory test

use and minimize false test outcomes of current and future diagnostic tests.