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Journal of Autoimmunity xxx (2014) 1e34

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Journal of Autoimmunity

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Review

Lyme disease: A rigorous review of diagnostic criteria and treatment

Andrea T. Borchers a, Carl L. Keen b, Arthur C. Huntley c, M. Eric Gershwin a, *

a Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, CA 95616, USAb Department of Nutrition, University of California at Davis, Davis, CA 95616, USAc Department of Dermatology, University of California at Davis, Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 8 September 2014Received in revised form15 September 2014Accepted 16 September 2014Available online xxx

Keywords:Lyme diseaseSpirochetesAutoimmunityDiagnostic criteria

Abbreviations: ACA, acrodermatitis chronica atroAV, atrioventricular; Bb, Borrelia burgdorferi; CDC, CenPrevention; CNS, central nervous system; CSF, cerebimmunoassay; DbpA, decorin-binding protein A; EM,arthritis; LNB, Lyme neuroborreliosis; PCR, polymeras* Corresponding author. Division of Rheumatology

nology, University of California at Davis, School of MDrive, Suite 6510, Davis, CA 95616, USA. Tel.: þ1 5304669.

E-mail address: megershwin@ucdavis.edu (M.E. G

http://dx.doi.org/10.1016/j.jaut.2014.09.0040896-8411/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: BorcheAutoimmunity (2014), http://dx.doi.org/10.1

a b s t r a c t

Lyme disease was originally identified in Lyme, Connecticut, based upon an unusual cluster of whatappeared to be patients with juvenile rheumatoid arthritis. It was subsequently identified as a newclinical entity originally called Lyme arthritis based on the observation that arthritis was a major clinicalfeature. However, Lyme arthritis is now called Lyme disease based upon the understanding that theclinical features include not only arthritis, but also potential cardiac, dermatologic and neurologicfindings. Lyme disease typically begins with an erythematous rash called erythema migrans (EM).Approximately 4e8% of patients develop cardiac, 11% develop neurologic and 45e60% of patientsmanifest arthritis. The disease is transmitted following exposure to a tick bite containing a spirochete in agenetically susceptible host. There is considerable data on spirochetes, including Borrelia burgdorferi (Bb),the original bacteria identified in this disease. Lyme disease, if an organism had not been identified,would be considered as a classic autoimmune disease and indeed the effector mechanisms are similar tomany human diseases manifest as loss of tolerance. The clinical diagnosis is highly likely based uponappropriate serology and clinical manifestations. However, the serologic features are often mis-interpreted and may have false positives if confirmatory laboratory testing is not performed. Antibioticsare routinely and typically used to treat patients with Lyme disease, but there is no evidence that pro-longed or recurrent treatment with antibiotics change the natural history of Lyme disease. Althoughthere are animal models of Lyme disease, there is no system that faithfully recapitulates the humandisease. Further research on the effector mechanisms that lead to pathology in some individuals shouldbe further explored to develop more specific therapy.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Lyme disease takes its name from the town of Lyme in Con-necticut, U.S.A., where an unusual cluster of cases with an initialdiagnosis of juvenile rheumatoid arthritis occurred in the mid1970s [1]. Closer examination of this community revealed that therecurrent attacks of arthritis in these patients appeared to be a newclinical entity, originally called Lyme arthritis (LA). After it became

phicans; AI, antibody index;ters for Disease Control andral spinal fluid; EIA, enzymeerythema migrans; LA, Lymee chain reaction., Allergy and Clinical Immu-edicine, 451 Health Sciences752 2884; fax: þ1 530 752

ershwin).

rs AT, et al., Lyme disease:016/j.jaut.2014.09.004

clear that arthritis constituted only one of the late manifestations ofa multisystem disease that included dermatological, neurologicaland cardiac manifestations [2], the name was changed to Lymedisease.

Early prospective studies in the US revealed the natural historyof this disease in untreated patients. Lyme often begins with anerythematous rash, consistent with an entity called erythema mi-grans (EM) or erythema chronicum migrans that had first beendescribed in 1909 in Europe [3]. Some patients (20%) experience nofurther signs and symptoms, the remainder may develop neuro-logical (11%), cardiac (4e8%) or rarely ocular abnormalities, and45e60% eventually develop arthritis [4,5]. Not only EM, but also ameningoradiculoneuritis had long been known to occur after tickbites in Europe and to be associated with each other [6]. In 1982Burgdorfer et al. [7] isolated a previously unidentified spirochetefrom deer ticks (Ixodes dammini, now Ixodes scapularis) (Fig. 1).Their and other data demonstrated that this was the etiologicalagent of Lyme disease [7e9], which was named Borrelia burgdorferi(Bb) in honor of one of its original discoverers [10]. These bacteria

A rigorous review of diagnostic criteria and treatment, Journal of

Fig. 1. Ixodes scapularis, the primary vector for Lyme disease in eastern North America.Photo credit: A new view on lyme disease: rodents hold the key to annual risk. Gross L,PLoS Biology Vol. 4/6/2006, e182. http://dx.doi.org/10.1371/journal.pbio.0040182(Creative Commons Attribution 2.5 Generic license).

A.T. Borchers et al. / Journal of Autoimmunity xxx (2014) 1e342

belong to the phylum Spirochetes, have an outer membrane sur-rounding a protoplasmic cylinder, are long (10e30 mm), but thin(0.18e0.25 mm in diameter), irregularly coiled, and highly motiledue to a 7e11 flagella inserted at each end of the cell and containedin the periplasmic space. They have a small linear chromosome anda variable number of linear and circular plasmids (up to 21 plas-mids, 12 of which are linear, while 9 are circular) [11]. The genomeis highly complex, with almost 8% of all open reading framesencoding lipoproteins and another 6% devoted to motility andchemotaxis. Since open reading frames in the Borrelia genomeoften lack significant homology with previously annotated genes,the function of a considerable portion of their products remains tobe elucidated. Interestingly, if an organism had not been identified,Lyme would be considered as a classic autoimmune disease withsimilarities to other human diseases with loss of tolerance andseemingly common effector mechanisms [12e15].

Spirochetes with similar morphology, protein profile and anti-genic determinants were detected in Ixodes ricinus ticks fromSwitzerland and Ixodes pacificus ticks from Oregon [7,16] and sub-sequently in Ixodes persulcatus ticks in Russia [17]. Genotypingsubsequently established that Lyme disease in Europe was causednot only by Bb sensu stricto, but also by two genotypically distinctspecies, which were named Borrelia afzelii and Borrelia garinii andsubsumed under the B. burgdorferi sensu lato complex (here Bor-relia species abbreviated as Borrelia spp.), which now contains atleast 20 confirmed or proposed genomic species (see Table 1). Ofthese, Borrelia spielmanii, Borrelia bavariensis, Borrelia bissettii,Borrelia lusitaniae, Borrelia americana, Borrelia andersonii and Bor-relia valaisiana also have pathogenic potential (see Table 1 for theavailable evidence). Of particular note, it had long been held that Bbis the only species causing Lyme disease in the US, but PCR-baseddetection of both B. americana and B. andersonii has been re-ported in blood or skin of a number of US patients, some of themwith EM and/or other manifestations of Lyme disease [18,19]. Inaddition, B. bissettii, which has been isolated from the cerebralspinal fluid (CSF) of a European patient with Lyme disease [20], hasalso been detected by PCR in serum of three Californian subjects,but details on whether this was associated with any disease man-ifestations are not available [21]. It should be kept in mind, how-ever, that detection by PCR or even isolation by culture of a

Please cite this article in press as: Borchers AT, et al., Lyme disease:Autoimmunity (2014), http://dx.doi.org/10.1016/j.jaut.2014.09.004

particular species of Borrelia does not prove its pathogenicity sinceit is not uncommon for patients to simultaneously harbor two ormore species [22e25]. While direct PCR of tissue samples appearsto be more sensitive than culture-based typing, it does not reliablyamplify all strains identified by culture [26].

Borrelia spp. are maintained in a complex enzootic cycleinvolving one or more vertebrate host reservoir species and one ormore tick species. The major vectors transmitting pathogenic Bor-relia spp. all belong to the hard-bodied ticks of the genus Ixodes,with I. scapularis transmitting Bb in the East, mid-Atlantic andupper Midwest of the US and I. pacificus transmitting Bb andB. bissettii in the Western parts of the US, whereas I. ricinus is themajor European vector for Bb, B. afzelii, and B. garinii along withother potentially pathogenic and non-pathogenic Borrelia spp., andI. persulcatus, whose geographical distribution partially overlapswith I. ricinus, is the main vector of B. afzelii and B. garinii in easternregions of Europe and in Asia. Note, however, that there have beenisolated reports implicating other tick genera in transmitting Bband other Borrelia spp., including Amblyomma americanum inFlorida and Georgia [18] and ticks of the genus Dermacentor inSpain [27]. The biology of Borrelia spp. is closely tied to the lifecycleof the tick vector. Ticks take one blood meal during each of thethree stages of their life cycle, i.e., as larvae, nymphs, and adults.There is little to no transovarial transmission of Borrelia spp.infection, therefore ticks become infected when larvae or nymphsfeed on an infected host, infection is thenmaintained transstadiallyand can be passed on to the host providing the next blood meal,including humans.

2. Epidemiology of Lyme disease

Almost all confirmed cases of Lyme disease have occurred in theNorthern hemisphere, the majority coming from the United Statesand Europe (including the European part of Russia), far fewer fromAsia, and some from Northern Africa [28]. In the US, Lyme diseasehas been a nationally notifiable disease since 1991 and the numberof reported cases rose steadily from 9908 in 1992 to a peak ofalmost 30,000 confirmed cases in 2009 [29]. Since then, the inci-dence appears to have stabilized below 25,000 confirmed casesannually. There are numerous indications that underreportingrepresents a considerable problem [30e32], and scientists from theUS Centers for Disease Control and Prevention (CDC) actually esti-mate that the true number of cases may be closer to 300,000 eachyear [33]. On the other hand, it is equally clear that there is aconsiderable over-diagnosis of Lyme disease based upon misinter-pretation of the serology and/or over-zealous interpretation ofinternet information [34].

The Northeast, mid-Atlantic region and upper Midwest are theprimary areas of endemicity, and just 10 states (Connecticut,Delaware, Massachusetts, Maryland, Minnesota, New Jersey, NewYork, Pennsylvania, Rhode Island and Wisconsin) account for �93%of annual cases. But evenwithin these endemic areas, the incidencevaries considerably, exceeding 500/105 in certain counties [35].Among the cases reported to the CDC, AfricaneAmericans aresignificantly underrepresented [36,37]. This may be due to a lowerrisk of exposure since African Americans more rarely live in highlyendemic rural areas. However, the results of a detailed analysis ofcases in an endemic area of Maryland revealed that area of resi-dence alone could not fully account for the different incidence ratesof African Americans compared to European Americans [36].Instead, a significantly lower rate of EM and an excess risk ofarthritis among African Americans suggested that under-recognition of EM e or unawareness of its clinical significance e

resulted in a disproportionate number of late manifestations in thispopulation. Unlike many other immune-mediated diseases, there is

A rigorous review of diagnostic criteria and treatment, Journal of

Table 1Borrelia spp., their vectors, and evidence for their pathogenicity (culture from human tissue, demonstration of their DNA in human tissue, pathogenicity in mice). (List of species and vectors based on [600,601]).

Borrelia species Vector Geographicaldistribution

Culturedfrom blood

Cultured from EM Isolated from other tissue

Positive PCR Pathogenicity in mice

B. afzelii I. ricinus, I. persulcatus,I. hexagonus

Asia, Europe Rarely [104,105] [20,111,167,168,391] Synovial fluid or tissue,CSF [20,24,110,113,385]ACA [395,396]BL [184]

CSF, blood [104,388];synovial fluid [602,603];heart valve [366]

[604]

B. americana I. pacificus, I. minor USA Blood or skin [18,19]B. andersonii I. dentatus USA Blood or skin [18,19]B. bavariensis

(formerly OspAserotype 4 ofB. garinii)

I. ricinus Europe [391] EM [412]

B. bissettiia I. ricinus, I. scapularis,I. pacificus, I. minor,I. affinis

Europe, USA [605] CSF [20,605];BL [184]

Serum [21]; blood [606];cardiactissue [607]

[608]

B. burgdorferi sensustricto

I. ricinus, I. scapularis,I. pacificus,I. spinipalpis, I. hexagonis,I. affinis, I. minor,I. muris

Europe, USA [101,103,405] [20,80,164,166] Synovial fluid, CSF[20,24,112,385]Heart [20]ACA [395,396]

CSF, blood [104,220,388];synovialfluid or tissue[118,268,384,578,602,603]

[379,380,609,610]

B. californiensis I. pacificus, I. jellisonii,I. spinipalpis

USA

B. carolinensis I. minor USAB. garinii I. ricinus, I. persulcatus,

I. nipponensis,I. uriae

Asia, Europe Rarely [105,110] [20,111,166,168] Synovial fluid or tissue, CSF[20,24,111,385]ACA [110,395,396]BL [394]

CSF, blood [104,388];synovial fluid [602,603]

[369,611,612]

B. finlandensis I. ricinusB. japonica I. ovatus Japan In inbred mice [613], but

not in outbred mice [614]B. kurtenbachiia I. scapularis, (I. ricinus) Europe, USA CSF or skin [605] Weak [615], absent [616];B. lusitaniae I. ricinus Europe, North Africa [617] Yes, but not arthritogenic

[611,618]B. sinica I. ovatus ChinaB. spielmanii I. ricinus Europe [20,145,382,619,620]B. tanukii I. tanuki JapanB. turdi I. turdus JapanB. valaisiana

(VS116)I. ricinus, I. granulatus,I. nipponensis,I. columnae

Asia, Europe Skin [23]; blood [621]; CSF[622]; B. valaisiana-relatedspeciesin blood [234]

[611]

B. yangtze I. granulatus, I. nipponensis,Haemaphysalis longicornis

China

Genomospecies 2 I. pacificus, I. spinipalpis USA

a B. bissettii and B. kurtenbachii (initially called Bb strain 25015) were originally both included in the B. bissettii genospecies. Therefore, it is not always possible in older studies to determine which patient isolates belong towhich of these novel species.

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no obvious sex bias (see below) nor is there any significant data onconcordance in twins or within large family pedigrees [38e40].

In most of Europe, Lyme disease is not a reportable disease, anduntil recently there were no standardized case definitions [41].Therefore, the available data are less reliable and difficult tocompare because a variety of active and passive surveillancemethods were used to arrive at the available estimates. Nonethe-less, it is clear that Lyme borreliosis is highly endemic in much ofEurope, with the highest incidence reported from southern Swe-den, Lithuania, Germany, Austria, and Slovenia. The total number ofannual cases in Europe is estimated to be ~3-fold higher than thenumber of the cases reported to the CDC [28]. A rise in the inci-dence of Lyme borreliosis similar to that observed in the USA hasbeen reported from some European regions [42e44]. Increasedawareness of the disease almost certainly accounts for some of thisrise, but climate change with a subsequent expansion of the terri-tory of vector ticks, along with changes in land use, resulting inincreases in reservoir hosts for ticks, and changes in residentialdistribution and human recreational behavior also contribute;there is also the potential error of a false positive diagnosis[42,45e49].

While the CDC data in the USA suggest that males constituteslightly more than half of the reported cases (54% overall and 61% ofpediatric cases) [37], data from Europe often suggest a slight tomarked female preponderance [28,42,50], but an even distributionhas been reported from some areas [51e53]. The age distributionamong cases with Lyme disease is generally bimodal, with thehighest incidence rates seen in children aged 5e9 years and inadults >50 years of age in both the US and Europe[28,35,37,42,51,54].

3. Risk of infection after a tick bite and factors influencing it

3.1. Risk of infection

In areas that are endemic for Lyme disease, the risk of Borreliainfection after the bite of an infected tick is only 1% and 3% in the US[55e57], and 3e12% in Europe [58e62]. However, at least one-fourth, and sometimes more than half, of European subjects sero-convert without developing clinical manifestations of Lyme disease[58e61]. In a cohort of highly exposed individuals, almost all of theseroconverters remained asymptomatic [63]. This is the moreremarkable since a substantial proportion (�30%) of European pa-tients with EM never develop seropositivity [64e66]. In contrast,asymptomatic seroconversion is quite rare in the US [67,68]; themechanism for this is unclear and further studies are suggested.

3.2. Environmental and behavioral factors

Risk factors for Borrelia spp. infection are time spent in endemicareas, time spent outdoors, and factors associated with tick density[69e71]. Therefore, the most effective way to minimize the risk ofBorrelia infection is to avoid tick habitats, such as wooded areas,shrubs, tall grass and particularly the edges where these differenttypes of vegetation meet. Wearing protective clothing, using DEETinsect repellents, conducting a visual inspection every night tocheck for attached ticks, and prompt removal of attached ticks areother important preventive measures. Of note, a previous diagnosisof Lyme disease was identified, along with age, as an independentpredictor of Bb infection in a study that investigated both envi-ronmental and behavioral risk factors [70]. This probably reflectsthat the same population subgroups stays at risk of repeated in-fections because of the same behavioral or environmental factorsthat increased their original risk. The role of environment versusheritability is well established in human immune diseases [72]. The

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high rates of reinfection observed in several cohort studies confirmthat a previous history of Lyme disease constitutes a risk factor forreinfection [73e75]. This also means that previous infection doesnot protect from reinfection. Mice do develop immunity, but it isstrain-specific [76], and there are recent empirical data suggestingthat humans also develop strain-specific immunity that lasts for�6years [77]. This could explain the extraordinarily low rate ofsymptomatic infection (~2%) in a Swiss cohort with frequentexposure to ticks most likely containing a wide variety of strains[63]. It is important to note that Lyme disease follows the pattern ofa classic infection, much like that of post-streptococcal glomeru-lonephritis or rheumatic heart disease. It is not related to trauma,exercise, automobile accidents, or weather. It is strictly a hostpathogen relationship. Indeed, both in the case of Lyme as well asany other human immunologically mediated disease, the role forenvironmental associated factors has reasonably defined criteria[78].

3.3. Season and tick developmental stage

In both the Eastern US and most parts of Europe, peak onset ofLyme disease occurs during the summer months, when nymphalabundance is highest and nymphs are actively questing, whereasfew cases occur during the late fall and the first few months of theyear when adult ticks actively seek a host [51,54,64,79,80]. How-ever, in some European regions, a secondary peak in the EM inci-dence is seen late in the fall (October, November), suggesting thatactively questing adults also contribute to human Borrelia in-fections [64,81], and in Russia, Borrelia spp. are transmitted almostexclusively by adult I. persulcatus ticks [62]. Infection rates arehigher in adult than in nymphal ticks [20,79,82], but nymphs aremore abundant and are much smaller and more difficult to detectand, consequently, are more likely to stay attached longer [59].Duration of tick attachment has been identified as an importantrisk factor for infection, at least in the US.

3.4. Duration of tick attachment

Spirochetes reside in the midgut of unfed ticks. Once the bloodmeal reaches the midgut, the spirochetes begin to replicate andthen disseminate via the hemocoel to the salivary glands [83].There are experimental data demonstrating that Bb-infectedI. scapularis nymphs transfer considerable numbers of spirochetesas early as 24 h after attachment [84]. Nonetheless, in experimentalanimals, infection rarely occurs within 24 h of tick attachment, andpeak infection rates are not reached until 48e72 h of attachment[85e90]. The time required for infection to occur appears to dependon the target host species [85,86] and on the infecting strain of Bb[88]. In accordance with these findings, the risk of infection forresidents of endemic areas of the US was found to be minimal ifticks stayed attached for <72 h and to increase significantly withlonger attachment duration [55,67].

Data on the transmission of Borrelia spp. by I. ricinus ticks arelimited and obtained in gerbils [91,92]. These data suggest thatconsiderable rates of host infectionmay be observed as early as 17 hpost-attachment [91] and that I. ricinus transmits B. afzelii soonerand more efficiently compared to Bb [92]. In accordance with theexperimental animal data, attachment time does not appear to be amajor factor in determining the risk of infection in Europe [59,61],and there are a number of reports of European subjects becominginfected after a tick attachment duration of �24 h [59,60,64],particularly in Russia [62]. It is often hypothesized that the earliertransmission of European Borrelia spp. may be attributable to ahigher rate of systemic infection in unfed I. ricinus nymphs,including infection of the salivary glands, but data in support of this

A rigorous review of diagnostic criteria and treatment, Journal of

Fig. 2. Erythema migrans e erythematous target-like plaque of Lyme disease. Theprimary skin lesion of Borrelia infection is noted for centrifugal expansion, sometimesleaving central clearing. Photo Credit: James Gathany Content Providers(s): CDC/JamesGathany e This media comes from the Centers for Disease Control and Prevention'sPublic Health Image Library (PHIL), with identification number #9875.

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hypothesis are limited [93e95] and the rates of systemic infectionof unfed I. scapularis nymphs in the US are quite similar to thosereported from Europe [96].

3.5. Inoculum size

The minimal infective dose in humans is unknown, but only lownumbers of spirochetes (20e60/gland) can be detected in salivaryglands of feeding ticks [83,89,97], suggesting that a small inoculumis sufficient to cause infection and disease. In mice (mostly C3H),the ID50 (infectious dose at which 50% of animals became infected)for subcutaneous needle inoculation of salivary gland extracts ob-tained from partially fed nymphal ticks was 18 spirochetes [98]. TheID50 values in syringe inoculation experiments are somewhathigher and greatly depend on the Bb strain under investigation[83,99,100].

4. Manifestations and diagnosis

4.1. Stages of Lyme disease

It is customary to divide Lyme disease into three stages. The“primary lesion” or early localized stage occurs at the site of Borreliainoculation. The infection is manifest as erythema migrans(migrating redness), an erythematous papule that expandscentrifugally over days to become a plaque, with or without centralclearing (Fig. 2). With the development of EM, the patient may alsohave constitutional symptoms such as fever, malaise, and headache.The skin lesion may have localized pain or pruritus, and regionallymphadenopathy may develop. In ~20% of untreated subjects, thisearly stage with EM remains the only manifestation of Lyme dis-ease. In the first few weeks after infection, spirochetes can becultured from blood of up to 45% of American patients, indicatingthat they disseminate via the bloodstream to other tissues[101e103]. Note, however, that culture isolation from blood ismuchless frequently achieved in European patients (0.7e7.7%)[104e106], possibly suggesting other routes of dissemination. The“secondary lesions” of Borrelia infection, the early disseminatedstage, comprises multiple EM lesions, widespread erythematousplaques that are smaller but morphologically similar to the initiallesion. The early disseminated stage may also be associated withLyme neuroborreliosis (LNB), and carditis. European patients mayalso develop a skin lesion called borrelial lymphocytoma duringthis stage (Fig. 3). The “tertiary skin lesion” of this infection, iden-tified for decades before the associationwith Borreliawas known, isacrodermatitis chronic atrophicans, a focal area of atrophic skinusually involving an extremity. Acordermatitis chronica atrophi-cans (ACA) is primarily found in Europe, and is most commonlyassociated with the late disseminated stage, which can also includelate neurological manifestations. Although LA can occur during theearly disseminated stage, it is more commonly observed during thelate disseminated stage.

4.2. Case definitions

There are no standardized diagnostic criteria for Lyme borre-liosis. Unfortunately, this has led to both over and under diagnosisof Lyme disease. Clearly the disease requires the tick, the spirocheteand the host. In other words, the presence of ticks by themselvesdoes not in any fashion lead to the diagnosis of Lyme disease. TheCDC has published case definitions for surveillance purposes (seeTable 2), but emphasizes that these are not intended as diagnosticcriteria [107]. The European Concerted Action on Lyme Borreliosis(EUCALB) has proposed clinical case definitions for use in clinicalsettings and epidemiological investigations (see Table 3) (EUCALB,

Please cite this article in press as: Borchers AT, et al., Lyme disease:Autoimmunity (2014), http://dx.doi.org/10.1016/j.jaut.2014.09.004

www.eucalb.com) [41]. Except for EM, the manifestations of Lymedisease are not specific, but can have a variety of causes. It istherefore vital to obtain a detailed history in order to establishprobable exposure to Ixodes ticks in an endemic area at an appro-priate time of year and to obtain appropriate and definitive labo-ratory confirmation. This is a point that should be stronglyemphasized to avoid misdiagnosis.

4.3. Laboratory confirmation

4.3.1. CultureWith the exception of EM, all manifestations of Lyme borreliosis

require laboratory confirmation. The diagnostic gold standard is theisolation of Borrelia spp. by culture with subsequent PCR-based orother confirmation of its identify. Since culture is expensive andrequires special media and laboratory expertise, it is not routinelyperformed particularly since results are not available for 2e6weeksand, therefore, are not useful for clinical decision making. In addi-tion, the sensitivity of the procedure is 40e70% for EM[64,80,108,109], but only ~3e17% for CSF samples [104,110,111], andvery low for synovial fluid or tissue samples [112,113]. Therefore,negative results do not exclude the diagnosis of Lyme borreliosis.

A rigorous review of diagnostic criteria and treatment, Journal of

Fig. 3. Borrelial lymphocytoma. Although these lesions more commonly occur oncooler areas such as ear lobes and scrotum, the may appear elsewhere including at theoriginal site of inoculation and present as erythematous to purplish discrete nodules.Photo credit: “Borrelial lymphocytoma” by Spectrum11 e Own work. Licensed underCreative Commons Zero, Public Domain Dedication via Wikimedia Commons e http://commons.wikimedia.org/wiki/File:Borrelial_lymphocytoma.jpg#mediaviewer/File:Borrelial_lymphocytoma.jpg.

Table 2CDC case definitions for surveillance purposes [107].

Case definitions CDC

Erythemamigrans

A skin lesion that typically begins as a red macule orpapule and expands over a period of days to weeks toform a large round lesion, often with partial centralclearing.the largest diameter must reach a size �5 cm;the diagnosis must be made by a physician;laboratory confirmation is recommended for personswith no known exposure.

Neuroborreliosis Any of the following, alone or in combination:lymphocytic meningitis;cranial neuritis, particularly facial palsy (may bebilateral);radiculoneuropathy;encephalomyelitis.Encephalomyelitis must be confirmed by Bb-specificantibody production in CSF

Musculoskeletalsystem

Recurrent brief attacks of objective joint swelling in oneor a few joints, sometimes followed by chronic arthritisin one or a few joints

Cardiovascularsystem

Acute onset of high-grade (2nd or 3rd degree)atrioventricular conduction defects that resolve in daysto weeks and are sometimes associated withmyocarditis

Suspected A case of EM without known exposure (defined ashaving been �30 days before the onset of EM inwooded, brushy, or grassy areas in a county in whichLyme disease is endemic)A casewith laboratory evidence of infection but withoutavailable clinical information

Probable Any other case of physician-diagnosed Lyme diseasethat has laboratory evidence of infection

Confirmed A case of EM with a known exposureA case of EM with laboratory evidence of infection andwithout a known exposureA case with at least one late manifestation that haslaboratory evidence of infection

Laboratoryevidence

Positive culture for Bb ORTwo-tier testing (for specific antibodies) interpretedusing established criteria, wherePositive IgM is sufficient only when �30 d fromsymptom onsetPositive IgG is sufficient at any point during illnessSingle-tier IgG immunoblot seropositivity usingestablished criteriaCSF antibody positive for Bb by EIA or IFA, when the titeris higher than it was in serum

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4.3.2. PCRPCR on DNA extracted from tissue or fluid specimens is useful

for the confirmation of Borrelia infection, particularly in the syno-vium of patients with LA and also in cases of diagnostic uncertainty,but is generally performed only for research purposes. A large va-riety of methods have been used, but none of these is standardizedand, accordingly, distinct methods yield rather divergent results[104]. Its sensitivity is 75e80% in EM [23,108,114], 15e30% in CSF[104,115,116], and 60e85% in synovial fluid samples [117,118].Again, negative findings do not exclude the diagnosis of Lymedisease.

4.3.3. SerologySingle serological tests yield false-positive results in subjects

with other spirochetal infections; spirochete infection of gums isvery common. Hence, both the CDC and European guidelinesstrongly recommend a two-tier approach. The two steps consist of asensitive enzyme immunoassay (EIA), or rarely an indirect immu-nofluorescence assay, followed by immunoblotting of samples thatare positive or indeterminate in the first step. The CDC has pub-lished recommendations on the number and types of IgM and IgGbands that have to be present in order to consider immunoblotresults positive [119]. These rules should not be applied to patientswho were infected in Europe since the existence of at least 3pathogenic species requires species-specific interpretations rules[120]. In addition, with the currently available serological tests, theBorrelia-specific antibody repertoire seems more restricted [121],fewer patients with EM alone ever develop seropositivity (�70%compared to 80e86%) [64e66,122,123], and subclinical infectionappears to be considerablymore common in European compared toUS patients [56e61,63,67,68]. All of these factors are likely to lowerthe sensitivity and specificity of serological tests in the diagnosis ofLyme disease acquired in Europe. Known positive and negativesamples must be included in all assays; this is critical.

Obtaining serological confirmation of infection is hampered by avariety of problems. Antibodies against Borrelia spp. are slow todevelop, with IgM generally not being detectable for the first 1e2

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weeks after infection and IgG often not emerging for 4e6 weeks[51,122e127]. In patients who are seronegative at presentation butin whom there is strong suspicion of Borrelia infection, it is advis-able to obtain evidence of seroconversion, preferably within 8e14days after presentation [123,126]. However, some patients withsolitary EM as their only manifestation may never seroconvert[122,123], particularly in Europe [64e66]. The rate of seropositivitycorrelates with the duration of symptoms before diagnosis andtreatment not only in samples taken at presentation[51,126,128,129], but also in samples obtained during follow-up,indicating that early antibiotic treatment may abrogate the devel-opment of seropositivity [66,130]. There is great variability in thespecific antigens recognized by individual sera and the extent towhich serum antibodies recognize homologous proteins from otherpathogenic Borrelia spp. or even from heterologous strains of thesame species [99,121,131e133].

Both IgM and IgG Borrelia-specific antibodies may persist foryears in some patients [123,126,134,135], which make it impossibleto distinguish between past and newly acquired infections based onseropositivity alone. The background positivity rate in the generalpopulation may be high in endemic areas, ranging from 5 to 8.4% inUS studies with two-tier testing [136e138], and from 3 to 15% in

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Table 3Case definitions from the European Union Concerted Action on Lyme Borreliosis (EUCALB, www.eucalb.com) [41]

Manifestation Clinical Essential laboratory evidence Supporting laboratory/clinical evidence

EM Expanding red or bluish-red patch(�5 cm in diameter),a with or withoutcentral clearing. Advancing edgetypically distinct, often intenselycolored, not markedly elevated

None Detection of Borrelia spp. by cultureand/or PCR from skin biopsy

Borrelial lymphocytoma(rare)

Painless bluish-red nodule or plaque,usually on ear lobe, ear helix, nipple orscrotum

Seroconversion or positive serologyb

Histology in unclear casesHistologyDetection of Borrelia spp. by cultureand/or PCR from skin biopsyRecent or concomitant EM

ACA Long-standing red or bluish-red lesions,usually on the extensor surfaces ofextremities. Initial doughy swelling.Lesions eventually become atrophic.Possible skin induration and fibroidnodules over bony prominences

High level of specific serum IgG HistologyDetection of Borrelia spp. by cultureand/or PCR from skin biopsy

LNB In adults mainly meningo-radiculitis,meningitis;rarely encephalitis, myelitis;very rarely cerebral vasculitis.In children mainly meningitis and facialpalsy

Pleocytosis and demonstration ofintrathecal specific antibody synthesisc

HistologyDetection of Borrelia spp. by cultureand/or PCR from CSFIntrathecal synthesis of total IgM and/orIgG and/or IgASpecific serum antibodiesRecent or concomitant EM

Lyme arthritis Recurrent attacks or persistingobjective joint swelling in one or a fewlarge joints. Alternative explanationsmust be excluded

Specific serum IgG antibodies; usuallyin high concentrations

Synovial fluid analysisDetection of Borrelia spp. by cultureand/or PCR from synovial fluid and/ortissue

Lyme carditis Acute onset of atrio-ventricular (IeIII)conduction disturbances, rhythmdisturbances, sometimes myocarditis orpancarditis.Alternative explanations must beexcluded

Specific serum antibodies Detection of Borrelia spp. by cultureand/or PCR from endomyocardialbiopsyRecent or concomitant EM and/orneurologic disorders

Ocular manifestations Conjunctivitis, uveitis, papillitis,episcleritis, keratitis

Specific serum antibodies Recent or concomitant Lyme borreliosismanifestationsDetection of Borrelia spp. by cultureand/or PCR from ocular fluid

a If <5 cm, a history of tick bite, a delay (after the tick bite) of �2 days and an expanding rash at the site of the tick bite is required.b Initial and follow-up samples should be tested in parallel in order to avoid changes by inter-assay variation.c In early cases intrathecally produced specific antibodies may still be absent.

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various European regions and some Chinese provinces [139e142].But seroprevalence rates �20% and even as high as 37% have beenreported in high-risk groups, such as forestry workers or orienteers,by single-tier testing [28,63,143]. In addition, even commerciallyavailable diagnostic systems are not standardized and their resultscan differ substantially [129,144,145]. Further (see below) there arefalse positive results and data must be critically evaluated.

First generation assays are based on Borrelia spp. sonicates, butthese are derived from cultured spirochetes, which do not expressmany of the immunogenic proteins that are elaborated in vivo.Second and third generation EIA and immunoblot assays use avariety of recombinant proteins that are expressed in the vertebratehost, such as decorin-binding protein A (DbpA), variable majorprotein-like sequence, expressed (VlsE), or BBK32, and alsorecombinant forms of homologous proteins from differentgenospecies, truncated proteins or synthetic peptides[120,133,144,146e151]. In a protein microarray study, sera frompatients with Lyme borreliosis react with a variety of antigens thathad not been identified by conventional methods, most likelybecause they are expressed only in vivo [132]. While these datawere obtained in patients with late Lyme disease, they offer thepotential of improving the sensitivity of serological assays in earlyLyme borreliosis, which may remain suboptimal even with thirdgeneration assays [129,150,152].

Immunoblots are subject not only to intra and interlaboratoryvariation but also to subjective interpretation [153]. Again, knownpositive and negative samples and appropriate use of dilutions is

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necessary. Since no single EIA has sufficient specificity to be usedalone, efforts are being made to find a combination of EIAs thatyields the same or higher sensitivity and specificity compared toconventional two-tier testing [150,152,154]. A promising candidatefor one of these assays is a 26-mer peptide from the sixth invariantregion (C6) of the VlsE lipoprotein [124,139,152,154]. However, thisassay still does not eliminate the problem of cross-reactivity withother spirochetal and viral antigens and autoantigens, at least inEuropean patients [139], but this contrasts with results from the US[155]. In addition, the sixth invariant region of VlsE may not be ashighly conserved among strains of the three major pathogenicBorrelia genospecies as the original studies suggested [156], leadingto false negative results in a subset of European patients [157].Nonetheless, there are data suggesting that a USwhole cell sonicateELISA followed by a C6 ELISA can be used to identify patients withLyme regardless of their travel history, i.e., even if they acquiredtheir infection in Europe [158]. In contrast, conventional US two-tiered testing has very poor sensitivity in infections acquired inEurope.

4.4. Erythema migrans

The earliest sign of Borrelia infection is EM in ~70% of the casesreported to the CDC [159],�90% in cohorts of pediatric and adult USpatients [160e162], and 70e95% in European epidemiologicalstudies [51,53,54,163]. Adults are most commonly bitten on thelower limbs, buttocks or groin, less frequently on the trunk or arms,

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and rarely on the head, whereas the bite is on the head or neck in20e40% of children [28,64,80,160,164]. Onset is most commonly inthe warm summer months, and is rare from December to March.EM begins as a red macule or papule and expands over a period ofdays to weeks into a large round or oval red or bluish-red lesionthat can reach a size of up to 73 cm [165,166]. Central clearing mayeventually leave an annular lesion or a rash with the bull's eyeappearance that is considered to be so typical of EM. Actually,however, central clearing is seen in only 19% of US patients ascompared to almost 80% of European patients according to a meta-analysis [165], confirming the results of direct comparisons be-tween US and European patients [166e169]. Occasionally, ulcera-tion or vesicles may occur at the center of the lesion [80,164].Pruritus is reported by 33 and 38% of US and European patients,respectively [165]. In about 21% of US patients, but only 12% ofEuropean patients, secondary EM lesions develop [165]. Lymph-adenopathy is observed in 13e22% of patients.

EM is frequently accompanied, sometimes preceded, by sys-temic symptoms, in particular fatigue, headache, stiff neck, myal-gias, arthralgias, fever, nausea and dysesthesia [165]. The frequencyof systemic symptoms overall and of most of the individualsymptoms is lower in European patients with EM caused mostly byB. afzelii and B. garinii compared to EM caused by Bb in the US. Inaddition, lesions caused by B. garinii grow faster and reach a largersize than those caused by B. afzelii [109,168]. They were also foundto cause local symptoms, such as itching, burning, or pain moreoften, be accompanied by myalgia and chills more frequently andbe associated with a higher seropositivity rate, even though pa-tients with B. garinii infection presented earlier after symptomonset [168].

EM was once considered to be virtually pathognomonic of Lymedisease. Actually, this no longer holds true, at least in certain areas ofthe United States where a Lyme-like illness referred to as southerntick-associated rash illness (STARI) causes skin lesions that areindistinguishable from EM [165]. It is transmitted by the lone startick (A. americanum), but the agent causing STARI remains un-identified. The situation is further complicated by the fact that therehave been reports of the isolation of Borrelia spp. from A. ameri-canum [18], and the range of this tick is expanding into areas whereLyme disease is endemic. Otherwise, EM is considered to be suffi-ciently distinct in appearance to allow its clinical diagnosis withoutany laboratory confirmation in a patient with the appropriateexposure history. Serological testing is not advised since many pa-tients have not yet developed anti-Borrelia antibodies by the timethey presentwith EM [122e126].While the CDC case definitions forsurveillance purposes state that a solitary lesion must reach at least5 cm in size [107], lesions smaller than 5 cm can be diagnosed as EMby experienced physicians according to European case definitions[41] (see also Table 3). When erythematous lesions develop duringtick attachment or within 24 h after tick removal they most likelyrepresent hypersensitivity reactions. A variety of rashes can mimicEM (see the excellent review by Tibbles and Edlow [165] for thefeatures that help distinguish them from EM).

4.5. Other cutaneous manifestations of LB

4.5.1. Acrodermatitis chronica atrophicans (ACA)ACA is a late cutaneous manifestation of Lyme borreliosis that

primarily affects middle-aged and elderly women [170,171]. It oc-curs in 1e7% of European patients with Lyme disease [51,52,54,172],but was not seen in any Russian [53] or Chinese patients [173], andonly rare cases have been described in the US [174,175]. Its onset isvery insidious, with the appearance of a bluish-red discoloration ofa skin area with doughy swelling usually on the sun-exposedextensor surfaces of the hands and feet. In the beginning, it is

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usually unilateral, often on the same extremity that had beenaffected by EM months to years previously, but only ~20% of pa-tients give a history of EM [171]. ACA may later become symmet-rical and eventually affect all extremities in some patients [176].The initial inflammation can persist for years, but eventually isreplaced by atrophy of the skin [177]. Unlike EM and borreliallymphocytoma, it does not resolve spontaneously. Almost 20% ofpatients with ACA exhibit fibrous nodules particularly around theelbow or, less frequently, on other bony prominences such as theknee; others (~7%) have band-like fibrous lesions over the ulna ortibia [178]. At least 30e40% of patients develop a sensory orsensorimotor polyneuropathy that is most pronounced in the ex-tremities with cutaneous involvement [176,179].

Patients with ACA are usually strongly IgG-positive to a largevariety of borrelial antigens [121,178]. The differential diagnosis ofACA depends on the stage of the lesion and includes venousinsufficiency, acrocyanosis, livedo reticularis, lymphedema, andchilblains [175]. Histopathology may be helpful in supporting thediagnosis. A prominent histopathological feature is telangiectasesin association with a mostly perivascular infiltrate consistingmainly of lymphocytes with a variable number of plasma cells andmacrophages [178,180e182]. This infiltrate can be band-like, patchyor diffuse.

4.5.2. Borrelial lymphocytomaBorrelial lymphocytoma, also called lymphocytoma cutis or

lymphadenosis benigna cutis, is a presenting manifestation in0.3e4.5% of European cases of Lyme borreliosis[51,52,54,163,172,183], but is rarely reported from the eastern partsof Europe [53], while some cases from China have been described[173]. Children are affected more often than adults [51,54]. This B-cell pseudolymphoma manifests as a solitary red to bluish-redplaque or nodule a few centimeters in diameter, which may bepainful to touch [177,184e186]. It usually develops within weeks tomonths after infection, sometimes in association with EM or othermanifestations of early disseminated disease, but occasionally hasalso been observed late in the course of Lyme borreliosis togetherwith ACA [184]. It shows a strong predilection for earlobes inchildren, and nipples or genital areas in adults, but can develop inother regions of the body. A majority of patients either recall a tickbite or develop EM in the vicinity of the affected ear lobe or nipple,but in others the borrelial lymphocytomamay develop at a distancefrom the causative tick bite [177,184,185].

Solitary lymphocytoma cutis can have a variety of causes,although Borrelia infection is the most frequent one in endemicareas. Therefore, in the absence of EM, serological confirmation isrequired. The vast majority of patients with borrelial lymphocy-toma has Borrelia-specific IgM and/or IgG antibodies at presenta-tion or seroconverts over the course of the ensuing weeks[184e186], but some remain seronegative throughout the acutephase and follow-up [185,186]. If patients are seronegative, histo-pathology may be required, which shows a dense, diffuse lym-phocytic infiltrate with plasma cells throughout the dermis, rarelyextending into the epidermis [186]. Frequently, though not invari-ably, there are prominent lymphoid follicles with germinal centersthat e unlike other B-cell pseudolymphomas e typically lack amantle zone [186]. If untreated, borrelial lymphocytoma can persistfor months. With appropriate antibiotic treatment resolutiongenerally occurs within 3e4 weeks, but may take considerablylonger when diagnosis and treatment are delayed [184,185].

4.6. Lyme neuroborreliosis (LNB)

LNB most commonly develops within a few days to 3 monthsafter infection and can be the presenting and the only

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manifestation of Lyme disease [28,187e189]. Therefore, the onset ofearly LNB occurs most frequently in June/July to October/November, depending on the latitude [187,188,190e192]. Approxi-mately 10e15% of patients with untreated EM will developneurological sequelae [5,193]. Note, however, that when patientsare closely monitored and their earliest manifestations of Lymedisease are rigorously treated, as in two large US vaccine trials, theproportion of cases presenting with LNB can be as low as 2% or less[162,194]. Among the cases of Lyme disease reported to the CDC,12e14% have LNB [35]. Generally, LNB is the presenting manifes-tation in 16e23% of European patients with Lyme disease[51,53,163,195], although considerably lower rates have been re-ported in some regions [52,54]. All of the available epidemiologicaldata show the highest rates of LNB in children, followed by adults�50 years of age [51,54,187,196]. A very consistent finding is amale:female ratio of 1.5:1 among European LNB patients[187,188,191,196,197].

The classical triad of neurological manifestations in Lyme bor-reliosis, as originally described in Europe before knowledge of theetiological agent [198,199], consists of lymphocytic meningitis,cranial neuritis and painful radiculoneuritis alone or in variouscombinations. Although any cranial nerve can be affected, facialpalsy is the most common cranial neuropathy. This triad has beenobserved in North American patients, where lymphocytic menin-gitis with headache and stiff neck is most prominent, oftenaccompanied by mild encephalitis, whereas radiculoneuritis is lesspronounced [200,201]. Cranial neuritis is seen in about one half ofcases. Of note, among the cases reported to the CDC between 2001and 2010, Bell's palsy represented 9%, radiculoneuropathy 4%, andmeningitis/encephalitis 1%, respectively [159,202]. In contrast, inEuropean LNB, severe radiculitic pain that frequently worsens atnight is a common presenting symptom and is eventually reportedby 70e90% of adult patients [172,187,188]. CSF lymphocytic pleo-cytosis is present in the vast majority of European patientsdescribed to date [172,187,188,196], and now is a diagnostic crite-rion for definite LNB [203]. However clinical signs of meningitis arerare in adult patients [187,188]. Cranial neuritis affects 40e50% ofadult patients [51,172,187,188,196], with more than 80% of thesepatients having facial palsy, which is bilateral in over one third ofadults [187,188,200,201]. Paresis of other peripheral nerves canaccompany cranial neuropathies or occur in isolation. In a Europeanstudy, most patients with EM and moderate to severe symptomssuggestive of neurologic involvement (such as headache, stiff neck,vertigo, memory or concentration problems, sleep disturbances, orparesthesias) did not have laboratory evidence of infection and/orinflammation of the central nervous system (CNS) [145]. On theother hand, radicular pain, meningeal signs, and peripheral facialpalsy along with larger EM size and seasonal occurrence (onsetfrom May till August) were significantly associated with the pres-ence of lymphocytic pleocytosis, which was in turn predictive ofmicrobiologically proven LNB. Symptoms of CNS involvement suchas myelitis or encephalitis are rare in early LNB [187,188].

The characteristics of LNB in children differ from those seen inadults, with less frequent and less severe radicular pain, morefrequent headache and clinical signs of meningitis, while the fre-quency of facial palsy is often higher, but bilateral facial palsy is rare[172,187,189,190,196,204,205]. As a result, facial palsy and menin-gitis are the most frequent clinical manifestations of childhood LNB[189,190,192,206,207]. Lyme disease can be the most commonidentifiable etiology of facial nerve palsy in children in someendemic areas of the US [208] as well as Europe [189,191,209],particularly duringMayeOctoberwhen the vastmajority of cases ofLNB present [190]. Importantly, LNB in children may be quiteinsidious with slight meningeal signs, but otherwise mostly non-specific signs and symptoms such as headache, intermittent low-

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grade fever, loss of appetite and weight loss [192,205]. Rarely,children with meningitis may develop raised intracranial pressureand present with the typical clinical picture of pseudotumor cerebri[189,210].

There are also late manifestations of LNB. An encephalopathythat arises months or evenmany years after the initial infection hasbeen described mainly in US patients many of whom had not beentreated or had received delayed or inadequate treatment [211,212].These patients had objective evidence of slight cognitive dysfunc-tion and memory impairment along with sleep disturbances andmood changes. Some had evidence of CNS infection, includingintrathecal Bb-specific antibody production, i.e., a positive anti-Bbantibody index (AI), increased CSF protein levels or, more rarely,CSF pleocytosis [211e213], and their disease may have representedmild forms of encephalomyelitis. However, most of the patientsdiagnosed with Lyme encephalopathy did not have evidence of CNSinflammation and it has been suggested that they had a “toxicmetabolic” encephalopathy similar to that seen in other viral,bacterial or autoimmune inflammatory states [214]. In addition,chronic distal axonal polyneuropathies, manifesting mainly asintermittent distal paresthesias, have been described in US patients[211,215]. A similar distal neuropathy is seen almost exclusively inconjunction with ACA in Europe [176,179,216]. Another late disor-der seen primarily in Europe is progressive encephalomyelitis,which manifests with cerebellar ataxia, cognitive deficits, spasticbladder paresis, and para and tetraparesis, quite frequently inconjunction with cranial nerve palsies [187,188,217]. In addition,rare cases of chronic meningoencephalitis, meningomyelitis, andradiculomyelitis have been described [187,188].

The clinical diagnosis of LNB is highly likely when a patientpresents with classical triad of lymphocytic meningitis, cranialneuritis and painful radiculoneuritis, or with one or more of thesemanifestations in combination with a well-documented EM. Bilat-eral facial palsy, as seen in a considerable portion of adult patientswith LNB, is rarely seen in facial palsy of other etiologies[187,188,200,201]. Nonetheless, the definite diagnosis requireslaboratory confirmation, particularly when the symptoms ofpossible LNB are less characteristic. In the US, serum anti-Bb anti-bodies are considered to be adequate for this purpose inmost cases.Patients with LNB are usually seropositive at presentation or se-roconvert during the ensuing weeks [187,218]. When the CNS isinvolved, CSF analysis should show lymphocytic pleocytosis,elevated protein levels, and a positive intrathecal AI. In the case ofLyme encephalomyelitis, demonstration of a positive intrathecalanti-Bb AI is necessary to meet the CDC case definitions for sur-veillance. There have been no recent cohort studies of LNB in theUS, much less any recent systematic analyses of the sensitivity andspecificity of intrathecal AI and other CSF results. Therefore, the roleof CSF analysis in the diagnosis and management of otherneurological manifestations of Lyme disease remains uncertain[214,219e221].

Some European investigators have expressed considerableskepticism regarding the broad spectrum of neurological manifes-tations that have been attributed to Bb in the US without stringentdocumentation of specific intrathecal antibody production or evenpleocytosis [187,222]. Evidence of a positive Borrelia-specific anti-body index has long been the “gold standard” for the diagnosis ofLNC in Europe. It may be detectable in some patients with signs andsymptoms of LNB who still lack detectable specific antibodies inserum [129,144,188,223]. Unlike Borrelia-specific serum antibodies,which are present in a sizable subset of the general population[136,138,139], a specific AI in CSF is less likely to represent anincidental finding, at least in patients without a prior history of LBN.However, it may still be absent very early in the disease course[187,188,223,224] and may continue for months or years after

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successful antibiotic therapy [225,226], and even for decades insome untreated patients [222]. Unfortunately, patients often seekalternative therapies, including herbals and a variety of over-the-counter medications; there is no basis for the use of these in pa-tients with Lyme disease [227].

The guidelines published by the European Federation ofNeurological Societies (EFNS) distinguish between definite andpossible LNB [203]. Cases of definite LNB have to fulfill thefollowing three criteria: 1) neurological symptoms suggestive ofLNB, 2) CSF pleocytosis, and 3) intrathecal production of Bb-specificantibodies in CSF. For possible LNB, two of these three criteria haveto be fulfilled; and if criterion 3) is lacking, Borrelia-specific anti-bodies have to be detectable in serum after a duration of 6 weeks.Patients with i) peripheral neuropathy, ii) a clinical diagnosis of ACAand iii) Borrelia-specific serum antibodies also fulfill the criteria fordefinite diagnosis.

Some cases of facial palsy or other neurological manifestationsof Lyme disease that occur in conjunctionwith, or following shortlyafter, physician-documented EM may lack evidence of specificintrathecal antibody production, pleocytosis or both [188]. Someclassify such cases as LNB [188,191,197], and this might help avoidlumbar punctures in children [172]. It can even be argued thatlumbar puncture is unnecessary in European patients because itsresults do not influence treatment decisions [221] since it has beenshown that European patients with LNB can be treated with thesame regimen of oral doxycycline as is used for EM alone [228].

4.7. Cardiac manifestations

Carditis is a manifestation of early disseminated disease, pre-senting within days and up to threemonths after the onset of EM orother signs and symptoms of early Lyme disease, but it can be thesole presenting feature [229e231]. While early prospective studiesfrom an endemic area in the US revealed cardiac abnormalities in4e8% of patients with Lyme disease some of whom were system-atically evaluated for cardiac involvement [4,5,174], the incidencerates are much lower in more recent adult and pediatric cohorts[160,164,232,233]. According to the CDC case definitions only high-grade (second or third degree) atrioventricular (AV) conductiondefects represent notifiable cases of Lyme carditis, and theserepresent �1% of cases of Lyme disease reported to the CDC from2001 to 2010 [159]. Even though the European case definitions ofLyme cardioborreliosis are more comprehensive (see Table 3), �1%of patients presented with carditis in epidemiological studies invarious endemic regions in Europe [51,53,54,163]. Similarly lowincidence rates of cardiac abnormalities have been reported fromNortheastern China [173,234]. Lyme carditis affects primarily sub-jects in their 20se40s, with men outnumbering women by at least3:1 [230], while only a moderate male preponderance wasobserved among pediatric patients [231]. Details of what has beencoined the autoimmune side of heart disease has been reviewedelsewhere [235,236].

The most commonmanifestation of Lyme carditis is acute onset,rapidly fluctuating AV conduction disturbances [229e231,237,238].Electrophysiological studies show that different levels of the AVconduction system can be affected, but most commonly the block isabove the bundle of His, frequently within the AV node [229,230].Among the cases reported to date, almost half progressed to com-plete heart block, at least 20% had second grade AV block, and atemporary pacemaker was required in about one-third of thesepatients, whereas implantation of a permanent pacemakerwas rare[230,231,237,239]. In addition, myocarditis, pericarditis, endo-carditis, pericardial effusion, prolongation of the corrected QT-interval, ST-T wave changes and, in rare cases, congestive heartfailure have been reported. While patients with Lyme carditis can

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be completely asymptomatic, patients with high-grade AV blockusually show symptoms such as light-headedness, syncope or nearsyncope, shortness of breath, palpitations, and/or chest pain.Among pediatric patients with early disseminated Lyme disease,the lack of cardiopulmonary symptoms was found to be highlyspecific (99%) for the absence of carditis [231]. AV conduction dis-turbances can fluctuate and worsen rapidly, and patients with first-degree block and a PR interval �300 ms are at increased risk ofprogression to complete heart block [229,231]. Because of thepotentially fatal course of Lyme carditis [240e243], such patientsneed to be hospitalized and closely monitored.

Lyme carditis should be suspected in younger subjects withsymptoms of cardiac involvement in the absence of other apparentrisk factors and with a plausible history of exposure to Ixodes ticks.Patients with carditis are usually seropositive at presentation orseroconvert soon thereafter [231,239,240].

The potential role of Borrelia spp. in dilated cardiomyopathyremains uncertain. In 1990, Stanek et al. [244] reported the isola-tion of Borrelia spirochetes from, and their visualization in, endo-myocardial biopsy tissue of a patient with dilated cardiomyopathywho was strongly seropositive for antibodies against Bb. Morerecently, 24% of endomyocardial biopsy specimens from 41 Czechpatients with recent-onset dilated cardiomyopathy yielded positivePCR results for Borrelia spp. compared to none of the controls withend-stage coronary artery disease [245]. However, this was notreplicated in Dutch patients, and conflicting results also have beenobtained regarding an increased seroprevalence of Borrelia infec-tion among patients with dilated cardiomyopathy [246].

4.8. Lyme arthritis

If left untreated, ~60% of patients with EM develop briefrecurrent episodes of arthritis, often preceded and accompanied byarthralgias and myalgias [4]. Patients with EM would be treatedwith antibiotics today and would rarely if ever develop arthritis.Therefore, patients diagnosed with LA today must come mainlyfrom those subsets of patients whose EM goes unnoticed or un-diagnosed or who do not develop EM or any other typical earlymanifestations of Lyme disease. Approximately one-fourth ofadults and between 16 and 37% of childrenwith LA recall a tick bite[172,247e250]. More importantly, almost 30% of adults give ahistory of EM [172], even in case series recruited within the lastdecade [247], while the proportion in children has ranged between4 and 18% in recent cohorts [247,251,252]. According to data from alarge vaccine trial in which participants were very closely moni-tored, 16% of patients with Bb infection have only systemic symp-toms (primarily headaches and arthralgias) [253] that would notlikely be recognized as Lyme disease by the patient or the treatingphysician. An additional 7e11% of the participants show asymp-tomatic seroconversion [68,254]. Even under the worst-case sce-nario that 60% of these two patient subgroups develop LA, onewould expect arthritis to be the presenting manifestation in only~16% of patients with Lyme disease in the US. Rather surprisingly,patients with LA continue to represent ~30% of the cases reportedto the CDC [159]. In contrast, the observed rate in large case seriesof adults and children with Lyme disease has been �6% [160,161].In other words, the majority of cases of LA in the US would seem tobe preventable. LA constitutes 4e8% of cases of Lyme borreliosis inEuropean epidemiological studies [51,53,54,163], although the re-sults of a Swedish study suggest that LA may be underdiagnosed[255]. While Lyme disease overall affects men and women withsimilar frequency, up to 75% of adults and 70% of children with LAare male not only in the US [4,247,248,250,251,256e258], but alsoin Europe [170,172,249,259,260].

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LA can develop within days or weeks after EM or other mani-festations of early Lyme disease in both adults and children, butusually is a late manifestation that appears months or even yearsafter infection in untreated patients [2,4,53,256,261]. Therefore,onset of LA can be observed throughout the year [247e249,259]. LAis characterized by brief, but recurrent, attacks of monoarticular orasymmetric oligoarticular swelling and pain that generally affectlarge joints, most commonly the knee, or episodes of migratorypolyarthritis involving large and small joints [4,249,256,258e265].In early series, the temporomandibular joints were also quitefrequently involved [1,4], but this has rarely been described sincethen. While the attacks of arthritis eventually subside in manyuntreated patients, some develop chronic synovitis and, in rarecases, have evidence of erosions and permanent joint destructionand dysfunction [4,265]. This erosive arthritis histopathologicallyresembles rheumatoid arthritis [266,267]. With the notableexception of a European pediatric cohort [249], the affected jointsare usually painful, but the pain is often surprisingly mild consid-ering that the effusions are frequently large [1,252,256,258]. Rangeof motion is limited in the majority of patients and a considerableportion of patients is unable to bear weight on the affectedjoint [248,249,252,264]. Approximately one third of patientspresent with fever or give a history of recent fever[247,248,251,252,259,262]. Fatigue and malaise are fairly commonsymptoms, but other systemic signs and symptoms are rare in pa-tients with LA [1,4].

In accordance with arthritis mostly being a late manifestation ofLyme borreliosis, patients with LA generally are strongly seroposi-tive for Borrelia-specific IgG [121,131,247,252,258]. Since the anti-body repertoire expands over time, such patients usually displayIgG reactivity with numerous Western blot bands [121,131,258].Occasionally, however, LA presents shortly after the onset of EM [4],and serology may still be negative [268]. In such cases, amplifica-tion of spirochetal DNA from joint fluid or tissue is up to 85% sen-sitive and invaluable in supporting the diagnosis [117,118,268],whereas culture of Borrelia spp. from synovial fluid or tissue hasvery rarely been successful [112,113].

Markers of inflammation are elevated in peripheral blood andsynovial fluid [248,249,252,263,269], but may be in the normalrange in up to ~20% of patients [160,258,262]. None of these find-ings are specific for LA. Radiographs of the affected joint(s) mayshow effusions and soft tissue swelling, but are otherwise normal[4,249,269]. The differential diagnosis of LA includes primarily ju-venile idiopathic arthritis and septic arthritis; in all cases there arecommon cytokine, chemokine and cognate receptor alterations[270]. The ability to differentiate between LA and septic arthritiswould spare patients with LA unnecessary surgical irrigation anddebridement. Unfortunately, attempts to find predictors haveyielded inconsistent results [248,252,264].

5. Treatment

5.1. Treatment guidelines

The manifestations of Lyme disease are routinely treated withantibiotics; therapy hastens the resolution and largely prevents thedevelopment of other disease manifestations. The classes of anti-biotics that have shown the greatest effectiveness against Borreliaspirochetes is b-lactams (in particular cephalosporins) tetracyclinesand, to a lesser extent, macrolides. The best treatment approach, inparticular the duration of therapy, is a matter of ongoing debate. Itis quite evident that not all patients, and most certainly not allspecies or strains of Borrelia [113] respond equally to the antibioticsmost commonly used in the treatment of Lyme disease. Based onthe available evidence from randomized controlled trials (RCTs),

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treatment recommendations have been published by the InfectiousDiseases Society of America (IDSA) [221], the American Academy ofPediatrics [271], and by a variety of national and supranationalassociations in Europe [203,272] (www.eucalb.com). As the com-parison between the guidelines published by the IDSA and theEUCALB in Table 4 illustrates, the approaches to therapy are largelysimilar on both sides of the Atlantic, but there are some minordifferences in the recommended dosage and treatment duration.Note that the International Lyme and Associated Diseases Societyhas also published guidelines for the management of Lyme disease[273]. These advocate more aggressive and longer treatmentcourses for patients with persistent symptoms or refractory dis-ease, but much of the terminology is poorly defined and the cited“evidence” is largely restricted to older studies that support such anapproach and ignores much of the other available evidence. Theresults of several RCTs and a large retrospective study do not showany benefit in extending the duration of treatment with doxycy-cline from 10 days to 15 or 20 days in patients with EM [274e276].Retrospectively obtained data suggest a similar conclusion foramoxicillin [276]. In another double-blind RCT, treatment of pa-tients with disseminated Lyme borreliosis with a 3-week course ofi.v. ceftriaxone followed by 100 days of amoxicillin did not improvethe outcome compared to a 3-week course of ceftriaxone alone[277]. These findings do not exclude that some patients may benefitfrom longer or more aggressive therapy, but the risk-benefit ratiohas to be carefully weighed for each individual patient.

5.2. Antimicrobial prophylaxis after a tick bite

Due to the low rate of infection after a tick bite, even if the tick isshown to be positive for Borrelia spp. [55e61], treatment of a tickbite before any symptoms occur is not generally recommended inEurope [272]. However, a randomized placebo-controlled trial inthe US demonstrated the effectiveness of single dose of 200 mgdoxycycline within 72 h after removal of an I. scapularis tick inpreventing the development of EM [67]. Other manifestations ofLyme disease were not observed in this study subjects. In Russia,treatment with 200 mg doxycycline for 3e5 days after the tick bitereduced the infection rate by a factor of 11 [62]. The IDSA does notrecommend routine use of prophylaxis but states that a single doseof doxycycline may be offered provided that 1) the tick can bereliably identified as I. scapularis and has been attached for �36 h,2) prophylaxis can be givenwithin 72 h of removal of the tick, 3) thelocal infection rate of ticks is �20% and 4) doxycycline is not con-traindicated [221].

5.3. Antibiotic-refractory LA

LA that persists for >3 months after initiation of recommendedtreatment regimens with either i.v. antibiotics for >4 weeks or oralantibiotics for >8 weeks has been referred to as treatment-resistantor antibiotic-refractory LA [278,279]. According to the IDSA, symp-tomatic treatment with non-steroidal anti-inflammatory drugs(NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), orintra-articular corticosteroids is recommended for such patients ifPCR of synovial fluid or tissue does not show any borrelial DNA[221]. Note, however, that PCR is not 100% sensitive, hence negativePCR results do not necessarily mean the absence of spirochetes.Patients who still do not improve may benefit from arthroscopicsynovectomy, but this is not consistently successful [249].

5.4. Monitoring of Lyme carditis

Because of the rarity of Lyme carditis, the role of antibiotics in itstreatment is not fully established, particularly since Lyme carditis

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Table 4Treatment recommendations by the IDSA and EUCALB (In the Daily dose recommendations by EUCALB, “same” indicates that the same dose as recommended by the IDSA).

Condition Compound Route Adults (A),children (C)

IDSA EUCALB

Daily dose Duration(in days)

Daily dose Duration(in days)

EM, BL Doxycycline Oral A 2 � 100 mg 14 (10e21) Same 14 (10e21)Ca 2 � 2 mg/kg Same

Amoxicillin Oral A 3 � 500 mg 14 (14e21) 3 � 500e1000 mg 14 (10e21)C 50 mg/kg in 3 divided doses 25e50 mg/kg

Cefuroxime axetil Oral A 2 � 500 mg 14 (14e21) Same 14 (10e21)C 30 mg/kg in 2 divided doses 30e40 mg/kg

Penicillin V Oral A 3 � 1.0e1.5 Mio 14 (10e21)C 0.1e0.15 Mio/kg

Azithromycin e not recommendedas first line therapyb

Oral A 2 � 500 mg 7e10 2 � 500 mg 1st day1 � 500 mg Next 4 days

C 20 mg/kg 1st day10 mg/kg Next 4 days

Multiple EM As for EM above As for neuroborreliosisLNB Ceftriaxone i.v. A 2 g 14 (10e28) Same 14 (10e30)

C 50e75 mg/kg 50e100 mg/kgPenicillin G i.v. A 18e24 Mio in 4 divided doses 20 Mio 14 (10e30)

C 0.2e0.4 Mio/kg in 4 divideddoses

0.25e0.5 Mio/kg

Cefotaxime i.v. A 2 g every 8 h “Also effective”C 150e200 mg/kg

(Doxycycline may be adequate)d oral 2 � 100e200 mg (10e28) 2 � 100 mg or 1 � 200 mg 21 (14e30)Arthritis Doxycycline Oral 2 � 100 mg 28 Same 21 (14e30)

Amoxicillin Oral A 3 � 500 mg 28 3 � 500e1000 mg 21 (14e30)C 50 mg/kg in 3 divided doses 25e50 mg/kg

Cefuroxime axetil Oral A 2 � 500 mg 28C 30 mg/kg in 2 divided doses

Ceftriaxone i.v. A 2 g 21 (14e30)C 50e100 mg/kg

ACA Ceftriaxone i.v. A 2 g 21 (14e30)C 50e100 mg/kg

Doxycycline Oral 2 � 100 mg 21 (14e30)Amoxicillin Oral A 3 � 500e1000 mg 21 (14e30)

C 25e50 mg/kgCardio-

borreliosisParenteral antibiotic such as ceftriaxonefor hospitalized patients (as in LNB);followed by an oral antibiotic forcompletion of therapy (as in EM)

As for arthritis

c In Europe, azithromycin is primarily considered as an alternative treatment for patients who should not take doxycycline and are allergic to penicillin, such as children andpregnant or breast-feeding women.

a Doxycycline is not recommended for children <8 years of age or pregnant or breastfeeding women.b Macrolide antibiotics are less effective than other antimicrobials and should be reserved for patients for whom amoxicillin, doxycycline and cefuroxime axetil are

contraindicated.d In a non-inferiority RCT, oral doxycycline was shown as effective as i.v. ceftriaxone for the treatment of European LNB [228], confirming the results of earlier open-label

studies [221], but it is not yet clear whether this extends to North American patients with neurological manifestations.

A.T. Borchers et al. / Journal of Autoimmunity xxx (2014) 1e3412

regresses within one to six weeks without antimicrobial therapy[229]. Nonetheless, antibiotic treatment is recommended becausespirochetes have been visualized in endomyocardial biopsy speci-mens with various staining methods [231,280] or detected by PCR[240,241] and because carditis usually occurs at an early stage ofLyme disease when antibiotic treatment can prevent other diseasemanifestation. Carditis is a potentially life-threatening manifesta-tion of Lyme disease, and several fatal cases have been reported[240e242], one in a patient coinfected with Bb and Babesia microti[243]. Therefore, and since AV conduction disturbances can fluc-tuate and worsen rapidly it is highly advisable to hospitalize andclosely monitor patients with second or third degree block or firstdegree block and a PR interval �300 ms.

6. Outcome

6.1. Erythema migrans (EM)

In RCTs, EM usually resolves within a few days or weeks afterinitiation of treatment, although the process may take up to 8weeks [275,281e285]. Non-specific signs and symptoms, such as

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arthralgias, myalgias, fatigue, headache, irritability, stiff neck andparesthesias, often last longer, but their number and severitysteadily decreases over the following months [66,275,281,286]. Byone year after initiation of treatment, between 70 and >90% of USpatients return to their pre-Lyme disease health status[80,282,283,287e289], while rates of 84e98% have been reportedfrom Europe [66,275,286,290,291]. The prognosis in pediatric pa-tients also seems to be very good [160,292]. Treatment failures dooccur, and while major extracutaneous manifestations of Lymedisease often develop during or shortly after treatment, some occurmonths after therapy [160,274,281,283e285,289,290].

6.2. Lyme neuroborreliosis (LNB)

In many cases, the signs and symptoms of LNB resolve sponta-neously [189,211,222,293]. However, antimicrobial therapy not onlyhastens the resolution of symptoms [294], but also preventsobjective and subjective sequelae of LNB and the development ofother major manifestations of Lyme disease [293]. Symptoms ofearly LNB start to improve within a few days after initiation ofantibiotic therapy, and some patients recover completely by the

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end of treatment [189,294e297]. However, in most cases diseaseresolution is gradual. In particular motor and sensory deficits maybe slow to improve [228,294,295,298]. Some patients may requireretreatment because of insufficient improvement or even wors-ening. Generally, between 70 and 85% of patients with LNB make acomplete recovery, usually within 6e12 months after initiation oftherapy [196,296e302], and this rate may reach 90% in patientswith facial palsy [303]. The results of long-term follow-up studiessuggest that there is little further improvement after 1 year[197,304].

Residual neurologic findings can be documented in 5e28% ofpatients �1 year after therapy, including remaining facial nervedysfunction, other pareses, radiculopathies, vision or hearing loss,ataxia and various other findings [197,298,300,304e306]. Whilethey are generally considered to be mild to moderate, more thanhalf of the LNB patients identified in a population-based study inSweden felt that the neurologic sequelae persisting 5 years after theacute illness restricted their daily activities and almost one fifth hadnot been able to resume their pre-Lyme disease occupation [197]. In26 Swedish patients with CNS involvement with a median follow-up of 6 months (1e18 months), four patients (15%) had permanentdisabilities that seriously affected their daily lives, namelysphincter dysfunction in 2, unilateral hearing loss in 1, and nearcomplete unilateral peripheral facial palsy in 1 [307]. Patients whohad had LNB 30months previously also reported significantly lowerhealth-related quality of life (HrQoL) as assessed by the physicaland mental component of the Short-Form 36 (SF-36) and scoredhigher on the Fatigue Severity Scale (FSS) compared to matchedcontrols [305]. Their depression and apathy scores were also higher,but both patients and controls had low scores overall. Predictors ofthe physical health (the physical component summary score ofthe SF-36) were pretreatment symptom duration >6 weeks andincomplete recovery at 4 months, while the mental componentsummary score was predicted by incomplete recovery at 4 months.Almost half of the variation in fatigue (assessed by the fatigueseverity scale) was explained by pretreatment symptom duration>6 weeks, higher pretreatment clinical scores and non-recovery at4 months [304]. Others similarly found an association betweenpretreatment symptom duration and persistence of symptomspost-treatment [196], but particularly of remaining objectiveneurologic sequelae [197,298].

The complete resolution rate in children with facial nerve palsyranges between 80 and 96%, depending to some extent on thethoroughness of the assessment and the length of follow-up[308e312]. Of note, among pediatric patients who had beentreated for Borrelia-associated facial palsy 3e5 years earlier and hadall declared themselves completely cured, approximately one-halfstill had subjective feelings of facial dysfunction and a similarproportion had objective evidence of mild to moderate facial nervedysfunction upon neurophysiological testing, but there was littlecorrelation between the two [311]. Such patients may have diffi-culties with closing their eyes or pronouncing words, cosmeticcomplaints and social problems [310,313]. More limited data areavailable on the long-term outcome of pediatric LNB overall andthese are highly variable, indicating between 8 and 27% of patientsdo not recover completely [197,204,313,314]. In addition to residualfacial nerve dysfunction, such children can have objective evidenceof other neurologic sequelae that can be sensory, motor or both incharacter [313]. Unlike adult LNB, pre-treatment symptom durationwas not associated with outcome in pediatric patients [204,313].

6.3. Lyme arthritis

LA eventually resolves spontaneously, but this may take years,with one child having been reported to have attacks of arthritis in

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the 7th and 8th year after onset [4,261]. Resolution can be hastenedconsiderably with antibiotic therapy [315]. The majority of patientswith LA require only a single course of antimicrobial therapy, butthis proportion varies between 48 and 90% for US adults [247,278]and between 52 and 95% for US children [247,250,251,256,258,316].Similar ranges have been reported from Europe [259,260,317].Resolution of LA usually occurs gradually over the course of severalmonths after a 4-week course of oral antibiotics [278]. Therefore, itis advisable towait for threemonths before retreatment [221]. Evenif not all physicians heed this recommendation, it is obvious thatsubstantial numbers of patients need to be re-treated because ofinsufficient response to a single course of antibiotics, and some ofthose patients fail to respond even to a second or third course ofantibiotics [249,278,316,318]. Such antibiotic-refractory LA (per-sisting for >3 months after initiation of recommended treatmentregimens) has been estimated to occur in approximately 10% ofadult US patients [278,319], but this proportion may actually beconsiderably higher [247]. Some data, including the results of arecent direct comparison between adults and children [247], sug-gest a more benign prognosis for pediatric LA in the US[250,258,262,263]. However, this is not confirmed in other in-vestigations [251,316], one of which found that 39% of pediatricpatients took >6 months and 13% took >12 months for diseaseresolution, and the last patient was free of arthritis after ~19months [316]. Unfortunately, there was very limited information onthe number of courses of antibiotics and other treatment modal-ities in these children. Similarly, LA is refractory to antibiotics in aconsiderable portion (up to 23%) of children and adolescent fromsome European countries, and 16% of patients still had ongoingarthritis 10 months to 1 year after initiation of therapy [249,320].Lyme arthritis eventually resolves in all patients, though it may takemonths or years and may require NSAIDs, DMARDs, intraarticularcorticosteroid injections, or synovectomy.

No predictor of antibiotic responsiveness could be identified inpediatric cohorts [251,316]. It remains uncertain whether intra-articular steroid therapy prior to antibiotic treatment is associatedwith a poorer response [259,278,321]. Cumulative duration of ep-isodes of arthritis, but not the overall pre-treatment symptomduration, was associated with non-responder status [278]. Inaddition, the HLA-DR4 specificity [278], particularly in conjunctionwith serological reactivity to the outer surface protein A (OspA)antigen [279], and more specifically HLA-DRB1*0401, DRB1*0101and related alleles have been associated with treatment-resistantLA [319]. There is also no available data on the role, if any, of epi-genetics in Lyme disease [322].

There are few data on residual symptoms after antibiotic ther-apy in patients with LA. In a prospective population-based study ofpediatric LA, none of the 90 patients had active arthritis a median of7 years after diagnosis, but four reported mild to moderateimpairment in school and sports activities due to continuingmusculoskeletal complaints [256] and such complaints have alsobeen reported to persist in ~8% of European patients [260,320].

6.4. Post-Lyme disease syndrome

In RCTs and prospective cohort studies, residual subjectivesymptoms after antibiotic therapy are reported by 3e27% of pa-tients with EM [75,80,160,274,281,283,289] and 10e40% of patientswith LNB [197,298,300,304e306,323]. Such information is largelylacking for patients with LA. The most common symptoms after EMalone are arthralgias, myalgias, fatigue, headache, irritability, stiffneck and paresthesias [197,298,300,304e306,323]. Problems withmemory and concentration may be other prominent symptoms insome patients with a history of Lyme disease [324e326], and thesealong with fatigue, pain and paresthesias also are the most

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A.T. Borchers et al. / Journal of Autoimmunity xxx (2014) 1e3414

common symptoms reported by patients with a history of LNB[197,298,304,327]. It should be kept in mind, however, that most ofthese symptoms are quite common in the general population.Conflicting results have been obtained in studies addressing thequestion of whether these symptoms are more frequent or moresevere in patients with a history of Lyme disease compared tohealthy controls. Compared to matched controls, no significantdifferences were seen in European patients with EM [275,286],Swedish children with a history of LNB [204,313], or in cohorts ofUS patients with a history of any type of Lyme disease [293,328]. Incontrast, others found a significantly higher frequency of subjectivesymptoms in pediatric and adult patients with a history of LNB[305,323,329] or of any manifestation of Lyme disease [324,330]and significantly poorer global health and functional status[305,324,330]. Thus, it currently remains unclear towhat extent thesymptoms persisting after antibiotic treatment are a direct or in-direct consequence of Lyme disease.When specifically asked, only aminority of patients consider Lyme disease to be the cause of theirsymptoms and difficulties with activities of daily living[81,328,329].

Patients with a history of LNB not only have subjective com-plaints of memory problems, but assessment of neuropsychologicalfunction �30 months after treatment revealed deficits particularlyin verbal memory, but also in visual memory, attention/executivefunction, and processing speed in adult patients compared tohealthy controls [302,306], though not in pediatric patients with ahistory of Lyme facial palsy [329]. In other patient cohorts, there isoften little correlation between subjective perception and objectiveneuropsychological evidence of memory deficits [325,330]. Yet,numerous studies have shown that a subset of these patients hasobjective evidence of memory impairment, particularly on verbaltasks, compared to healthy controls or patients who recoveredcompletely [324,325,331e336]. However, this is not an entirelyconsistent finding [293,330,337,338] and comparison to diseasecontrols has also yielded conflicting results [324,325,332e334,336].A variety of other domains are affected less consistently. In contrastto adults, adolescents exhibited greater impairment of visualmemory than of verbal memory [339].

The subjective symptoms and objective cognitive deficits per-sisting in some patients with Lyme borreliosis resemble what hasbeen described in untreated or inadequately treated US patientswho developed a subtle encephalopathy years after the onset oftheir disease [211,212,333,340,341]. Only some of these patientshad evidence of CNS infection or inflammation such as intrathecalantibody production or elevated CSF protein concentrations[211,213,340,342]. Kaplan et al. [343] considered such patients tohave encephalopathy if they had abnormal CSF results or a positivePCR reaction for Bb DNA, and to have post-Lyme disease syndrome(PLDS) if CSF analysis yielded normal results. Since then, the defi-nition of PLDS (or post-treatment Lyme disease syndrome or“chronic Lyme disease”) has been expanded considerably and theseterms are now used to describe patients who have any kind ofpersistent symptoms after what is considered adequate antibiotictherapy for their manifestation(s) of Lyme disease. Here again arigorous evaluation is indicated to be certain there are not othercauses, i.e., to avoid mislabeling a patient as having Lyme disease.

Unfortunately, there is no standardized case definition for PLDS,and this may account for some of the discrepant results obtained instudies of cognitive function after Lyme disease. The IDSA hasproposed such a definition, which requires 1) a documentedepisode of early or late Lyme disease fulfilling the CDC case defi-nitions for surveillance purposes, 2) resolution or stabilization ofthe objective manifestations of Lyme disease following a generallyaccepted treatment regimen, and 3) onset of symptoms within 6months of the diagnosis of Lyme disease and persistence of

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continuous or recurrent subjective symptoms of fatigue, wide-spread musculoskeletal pain, or complaints of cognitive difficultiesfor �6 months after completion of antibiotic therapy [221]. Afurther requirement is that these symptoms are of sufficientseverity to result in a decline of physical and/or social functioning.There are also a variety of exclusion criteria intended to insure thatthe patient does not have coinfections, objective manifestations ofLyme disease or other undiagnosed or previously diagnosed un-derlying diseases or conditions that would explain the patient'scomplaints. The challenge will be to find, and agree upon, the toolsfor assessing symptoms and their functional impact before thesecase definitions can be considered useful in unraveling the inci-dence, nature, and pathogenesis of PLDS [344].

6.4.1. Treatment of PLDSThere have been several randomized, double-masked, placebo-

controlled trials of antibiotic retreatment in patients with persis-tent symptoms after what is considered appropriate therapy fortheir manifestations of Lyme disease [326,336,345,346]. The in-clusion criteria and other basic characteristics of the three trialsthat used i.v. ceftriaxone for the retreatment and included anintention-to-treat analysis are summarized in Table 5. In the studyby Krupp et al. [345], the improvement in fatigue was similar in thetwo groups at 1 month, but sustained at 6 months only in thosepatients who received i.v. ceftriaxone, resulting in a significantdifference at 6 months. Cognitive function was mildly impairedcompared to normative data in both groups andwas not affected bytreatment. Fallon et al. [336] found that the ceftriaxone group hadsignificantly greater generalized improvement in cognitive func-tion at 12 weeks, but this was not sustained at 24 week. In patientswith greater symptom severity at baseline, ceftriaxone was asso-ciated with significantly greater improvement in physical func-tioning and current pain at 12 and 24 weeks, and in fatigue at 12weeks only. The effectiveness of antibiotic retreatment for fatigueseen in the Krupp study [345] was confirmed in a post-hoc analysisof the Fallon data [336,347]. There was no significant treatmenteffect on any of the outcome measures in the trials conducted byKlempner et al. [326,338]. In all three studies, treatment wasassociated with several serious or even life-threatening adverseevents.

Based on these results, several guidelines contain the conclusionthat further antibiotic therapy of patients with subjective symp-toms that persist for �6 months after treatment with recom-mended antibiotic regimens is “not useful” [221] “ineffective”[203], or has “not shown sustained benefit” [272]. These state-ments are false as written since two of these trials did show asignificant and sustained benefit in at least a subset of patients[336,345]. They also bring up the question of what the precise ex-pectations were from repeated antimicrobial treatment in patientswho had already been treated with multiple and prolonged coursesof antibiotics. Did anybody truly expect yet another course of cef-triaxone would cure patients who had failed to respond toextended antibiotic therapy? Be that as it may, the interpretation ofthese results and the question of whether or not patients withdisabling subjective symptoms should be retreated with (yetanother course of) antibiotics remains the subject of intense con-troversy [347e350]. What seems to be lacking from this discussionis the well-known fact that antibiotic therapy can induce consid-erable disturbances of the intestinal microflora. Indeed, there hasbeen no study of the microbiome in patients with Lyme diseasedespite increased evidence of the importance of the microbiome inimmune mediated pathology [351,352]. An intact microflora hasimportant functions in preventing the colonization of the gastro-intestinal tract by pathogenic bacteria and in providing continuedstimulation to the gut-associated immune system and thereby

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Table 5Characteristics of three placebo-controlled randomized controlled trials of antibiotic retreatment in PLDS.

Reference Krupp et al. [345] Fallon et al. [336] Klempner et al. [326,338] Klempner et al. [326]

n 55 37 Patients, 20 healthycontrols

78 (Seropositive) 51 (Seronegative)

Inclusion criteria 1) Age 18e70 years2) History of physician-

documented EM- or CDC-defined latemanifestations of Lymedisease confirmed bypositive ELISA andWestern blot serology

3) Completion (6 monthsbefore study entry) ofstandard antibiotic ther-apy for Lyme disease

4) Current severe fatigue(score �4.0 on a modifiedversion of FFS-11)

1) Age 18e65 years2) History of physician-

documented EM- or CDC-defined manifestations ofLyme disease and apositive or equivocalELISA confirmed byWestern blot serology

3) Current positive IgGWestern blot

4) Treatment for Lyme dis-ease with �3 weeks of i.v.ceftriaxone completed �4months before study entry

5) Subjective memoryimpairment that startedafter the onset of Lymedisease

6) Objective evidence ofmemory impairment (asassessed with the Wechs-ler Memory Scale-III)

1) Age �18 years;2) History of acute Lyme

disease acquired in the USand;a) History of single or

multiple EM, earlyneurologic or cardiacsymptoms attributedto Lyme disease, radi-culoneuropathy, or LA

3) Current positive IgGWestern blot;

4) Physician-documentedtreatment of acute Lymedisease with a recom-mended antibioticregimen;

5) Onset of chronic symp-tomsa within 6 months ofthe initial infection withBb and their persistencefor �6 months but �12years

As in seropositive patientsbut with a history ofphysician-documented EMand current negative IgGWestern blot

Treatment drug i.v. ceftriaxone 2 g/d orplacebo

i.v. ceftriaxone 2 g/d orplacebo

i.v. ceftriaxone 2 g/d for 30days followed by oraldoxycycline 200 mg/d for 60days or placebo

Same as seronegative

Treatment duration 28 days 10 weeks 30 þ 60 ¼ 90 days Same as seronegativeOutcome measures 1) Change in fatigue;

2) Change in mental speed;3) Clearance of Borrelia (as

assessed by OspA antigenin CSF)

1) Neurocognitiveperformance;

2) Physical outcome

1) Health-related quality oflife;

2) Cognitive function

Same as seronegative

Days of prior antibiotictherapy (mean except inKlempner trials)

A 44 ± 22 daysP 57 ± 64 days

An i.v. 76 ± 61A oral 240 ± 310P i.v. 58 ± 40P oral 179 ± 231

A median 71 (IQR 42e113)P median 60 (IQR 42e98)

A median 57 (IQR 33e120)P median 49 (IQR 31e83)

A ¼ antibiotic group, An i.v. ¼ previous i.v. antibiotic treatment received by the antibiotic group, A oral ¼ previous oral antibiotic treatment received by the antibiotic group.P ¼ placebo group.

a These self-reported symptoms included widespread musculoskeletal pain, cognitive impairment, radicular pain, paresthesias, dysesthesias, or profound fatigue andinterfered with the patient's functioning.

A.T. Borchers et al. / Journal of Autoimmunity xxx (2014) 1e34 15

shaping the responses of the overall immune system [353].Therefore, it may be time to finally consider the role of repeated andprolonged antibiotic treatment itself in the symptoms of patientswith PLDS.

7. Pathogenesis

7.1. Animal models

Elucidation of the pathogenesis of Lyme disease is hampered bythe lack of an animal model that reproduces all the facets of humanLyme disease. The relative absence of appropriate models likelyreflects the disparity that exists between genetic susceptibility andenvironmental factors [354]. The rhesus macaque develops most ofthe manifestations seen in humans, including EM and peripheralneuropathy [355], but is not widely available and difficult andcostly to handle. Specific-pathogen-free beagle dogs with tick-transmitted Bb infection represent the most faithful model of hu-man LA because they develop recurrent episodes of mono or oli-goarticular arthritis (lameness) after an incubation period of �2months [356e358]. Rabbits are one of the few species that developEM after Bb infection but they eventually clear the infection, whileuntreated patients appear to remain persistently infected

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[315,359,360]. A variety of smaller animals, including rats, hamster,and gerbils, have also been used. The most widely employed modelis various strains of inbred laboratory mice, but while susceptiblestrains develop arthritis and carditis, neurological manifestationsand EM are absent. Arthritis develops early after infection, resolvesspontaneously, and does not show the typical recurrent attacksseen in human patients, with the exception of mice deficient inCD28 [361]. In addition, the infiltrate in murine carditis is domi-nated by macrophages with only scattered lymphocytes[362e364], whereas lymphocytes are the major constituents of thehuman cardiac infiltrate [241,280,365], even if a case of B. afzelii-associated endocarditis with an infiltrate consisting mostly ofmacrophages, neutrophils and eosinophils has been reported [366].

Mice are most commonly infected by syringe inoculation of oneof a few select cultured strains of Bb, but cultured spirochetes ex-press a different protein profile than spirochetes transmitted byticks and individual strains of Bb differ in their pathogenicity[367e369]. In addition, syringe inoculation of cultured spirochetesneglects the suppressive effects of tick saliva on the host immuneresponse. Yet, this effect is substantial, as evidenced by the pro-found alterations in the cytokine profile of mice infested by unin-fected ticks compared to normal mice [370], the decreasedleukocyte recruitment to the skin and the increased spirochete

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Table 6Common methods for typing Borrelia spp. strains.

Loci Method Resulting groups

Intergenic spacer 16Se23S (rrserrlA)

PCR-based RFLPanalysis

Ribosomal spacer type(RST) 1e3

Intergenic spacer 5Se23S (rrferrlB)

PCR þ sequencing orPCR-based RFLPanalysis

Intergenic spacer (IGS)types

OspA (plasmid-borne) Monoclonal antibodies Serotypes 1e8ospC (plasmid-borne) PCR þ (Cold) single-

strand conformationalpolymorphism (SSCP)analysis;PCR þ Reverse line blot

Based on <2% or >8%sequence divergence,�70 groups designatedby combinations ofletters for the Borreliaspp. and numbers

A.T. Borchers et al. / Journal of Autoimmunity xxx (2014) 1e3416

burden in various tissues of mice co-inoculated with tick salivarygland extract and Borrelia spp. compared to Borrelia spp. infectionalone [371e373], and by major differences in the immune responseof mice with tick-transmitted infection compared to mice infectedvia needle inoculation [374e376]. In vitro, tick saliva or salivarygland extract can inhibit the functions of DCs, macrophages, neu-trophils and NK cells, and suppress T cell responses and humoralimmunity [377]. NK and other regulatory cells have been shown toplay a key role in autoimmunity [352]. Mice, dogs andmonkeys canbe inoculated with Borrelia spp. via nymphs that were infectedeither by capillary feeding or by placement on mice that had beeninfected by syringe inoculation [357,378e381]. Arthritis andmyocarditis develop later after infection and persist longer in tick-inoculated mice compared to needle-inoculated mice [378e380].Cystitis is seen in mice with tick-borne infection, but is rarely re-ported from needle-inoculated mice, even though their bladderalso frequently harbors high spirochete numbers [369,382]. Adrawback of this model is that the size of the inoculum cannot bestandardized.

7.2. Factors that influence pathogenesis

7.2.1. Organotropism of the different Borrelia genospeciesAs the data summarized in Table 1 show, each of the three major

pathogenic species of Borrelia can cause essentially all of the typicalmanifestations of Lyme disease. Nonetheless, there are numerousdata suggesting that each of them is associatedwith distinct clinicalmanifestations. Thus Bb shows tropism for the joint [383,384], asalso suggested by the apparently higher incidence of LA in the US,where Bb is essentially the only genospecies causing Lyme borre-liosis, compared to Europe, where Bb is relatively uncommon.B. garinii is most strongly associated with LNB [111,145,385e390],and B. afzelii appears to be overrepresented among skin isolates[20,23,24,111,390e392], in particular ACA [111,393e396].

However, there are some major shortcomings in the currentlyavailable data. Determination of the infecting species was based oncultured isolates in many of these studies, but culture of Borreliaspp. is not very sensitive, particularly in CSF samples[104,110,111,397], and even less so in synovial fluid or tissue sam-ples [112,113]. PCR-based amplification of DNA isolated directlyfrom tissue specimens generally shows greater sensitivity [114], butlargely depends on the specific PCR target [23,104], still lackssensitivity, particularly in CSF [104,115,116], and does not neces-sarily detect Borrelia spp. in all samples that are culture positive[104,108]. Furthermore, since patients with EM are now routinelytreated with antibiotics, determination of the infecting species ofBorrelia in samples from extracutaneous lesions is likely to yieldresults that are skewed towards those strains that do not also causeeasily recognizable EM lesions. Few of these studies examinedwhatis likely to be a major determinant of the probability of acquiring aninfectionwith a particular species of Borrelia, namely its prevalencein regional ticks [20,24,111,386,391,398]. The results are somewhatconflicting, most likely because of the small number of ticks and/orhuman samples examined in some of these investigations. Inaddition, tick and human samples were collected during differenttime periods and in different locations, thereby not taking intoconsideration that there may be considerable temporal andregional variation in the number of infected ticks and the distri-bution of Borrelia spp. in ticks [20,71,399,400]. Overall, the resultssuggest that B. garinii may be underrepresented in skin rather thanoverrepresented in CSF [20,24,386,391]. Note, however, thatB. afzelii infection of the nervous system may simply be under-recognized because it does not cause the typical clinical picture ofEuropean LNB associated with B. garinii infection, namely radi-culoneuritis and/or peripheral facial palsy [110]. Instead, detection

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of B. afzelii in CSF was associated with mostly nonspecific symp-toms, and most of the patients from whose CSF B. afzelii could becultured did not fulfill the European diagnostic criteria for LNB.

7.2.2. Virulence of the infecting Borrelia strainA variety of typing tools have been developed for the study of

spirochete evolution and phylogenetics. The ones most frequentlyused for classifying strains of Borrelia species into subtypes arelisted in Table 6. The biodiversity of Borrelia spp. subtypes decreasesfrom ticks to EM lesions and further to sites of disseminated disease[391,401]. In other words, ospC, RST, or other subtypes of Bb,B. afzelii and B. garinii differ in their infectivity and ability to causedisseminated disease [26,402e408]. This may be partly mediatedby differential abilities to induce pro-inflammatory cytokines[409]; the mechanisms remain enigmatic and recent technologies,including use of microRNAs, have not yet been studied [236].Nonetheless, the available data remain somewhat controversial[410e412]. One of the reasons for these discrepancies may be theuse of spirochetemia as a marker of dissemination. Yet, the rela-tionship between hematogenous spread and disseminated diseasemay be more complex than generally appreciated. This is high-lighted by the findings that even non-disseminating strains cancause transient spirochetemia in mice [413], and that hematoge-nous dissemination is not necessarily a sign of greater pathoge-nicity, at least in European patients with Lyme disease [65,105].

This may also explain why experimental models have failed toconsistently reproduce an association between specific RST typesand disseminated disease, even though individual RST types differin their pathogenicity [367e369,380]. It is also possible that thefactors determining dissemination differ between humans andlaboratory mice or that the RST classification does not have suffi-cient discriminatory power to predict the ability to disseminate andto cause severe disease manifestations. Typing of ospC alone mayalso be insufficient to capture the capacity for causing disseminatedinfection, since multilocus sequencing allows grouping of Bb iso-lates into clonal complexes (many of which contain more than oneospC type) that better predict the propensity for dissemination[414]. Thus, the association between ospC major groups and inva-sive disease may be due to strong linkage disequilibrium ratherthan a direct effect of OspC [414]. This is, however, in contrast todata suggesting that ospC type is related to disseminated diseasebecause invasive and noninvasive ospC types differ in their affinityfor plasminogen, which is activated to plasmin, a serine proteasethat degrades extracellular matrix and, thereby, enables bacterialdissemination [413].

It also should be kept in mind that detection by PCR or evenisolation by culture of a particular Borrelia genotype does not proveits pathogenicity since patients may harbor more than one strain[26,404,405,415]. In addition, almost all of the typing studies were

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performed on culture isolates, but culture-based genotype analysisunderestimates the genetic diversity of the infecting spirochetes.This was most clearly demonstrated by the detection of mixtures ofgenotypes in 43% of samples that were typed directly in tissuesamples compared to barely 6% of cultured samples [26]. In otherwords, isolates may be selected for their ability to grow in culturerather than for their pathogenic potential. On the other hand, dif-ferences in the results from culture-based and direct typing ofpaired patient specimens indicate that the direct method also doesnot reflect the full diversity of the infectious strains [26].

7.2.3. Host genetic factorsNone of the above studies take host factors into account. Yet,

individual strains of mice clearly differ in their susceptibility to thedevelopment of arthritis and carditis [416e419]. These and otherdata underscore that pathology is at least partly determined by hostgenetic factors in mice [420e423]. While mice with most H-2haplotypes develop moderate to severe arthritis, the H-2d haplo-type was associated with resistance [417]. However, there is evi-dence to suggest that murine susceptibility or resistance to arthritisis not attributable to the MHC itself, but is determined by genesoutside the MHC [421e425]. Rhesus macaques from separate col-onies differ in the frequency with which they develop certainmanifestations of Lyme disease, such as myositis or peripheralneuritis [426]. This suggests that in this outbred animal model,genetic host factors also play a role.

There are indications that the differential ability for rapidspirochete clearance, thereby limiting the pathogen burden in thejoint, is an important and probably genetically determined factor indecreasing the susceptibility for Bb-induced arthritis in certainmouse strains after infection via syringe [378,420,421,427] or tickbite [428]. Yet, other protective mechanisms must be in play inC57BL/6 mice (B6), which develop only mild arthritis despitespirochete numbers in the joint similar to those seen in arthritis-susceptible C3H mice [421]. Conversely, IL-10-deficient mice havea significantly lower tissue spirochete burden, yet develop moresevere arthritis than their wild-type counterparts [429]. In US pa-tients, no association was detected between the number of Bbspirochetes in EM biopsy samples and the number or severity ofsystemic symptoms [108]. Actually, the spirochete burden wasinversely associated with the size and the duration of the EM le-sion(s) [108], which have been shown to be directly correlated[164]. Therefore, these results suggest that there is a reduction ofthe number of spirochetes in the skin near the tick bite over time[108]. This could be a simple dilution effect as spirochetes spreadover a larger area of skin, but could also reflect immune-mediatedelimination of spirochetes or their dissemination into the bloodstream. In contrast, higher spirochete load (mostly of B. afzelii) inskin biopsy specimens was associated with the presence of sys-temic symptoms and of central clearing in the EM lesion of Euro-pean patients [114].

7.2.4. CoinfectionsIxodes ticks are vectors for a variety of infectious organisms

other than Borrelia spp. In North America, these include Anaplasmaphagocytophilum, the cause of human granulocytic anaplasmosis(HGA, formerly human granulocytic ehrlichiosis), B. microti, themain cause of babesiosis, and the relapsing fever spirochete Borreliamiyamotoi. These can also be transmitted by I. ricinus andI. persulcatus ticks in Europe and Asia, which additionally can carrythe flavivirus that causes tick-borne encephalitis. The existence ofsimultaneous infectionwith Bb and A. phagocytophilum or B. microtihas been firmly established by their cultivation from patient tissueswith subsequent PCR-based confirmation of their identity[430,431]. Based on these and serological methods, the reported

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rates of human coinfection with Bb and A. phagocytophilum or B.microti vary between 2e11% and 2e40%, respectively, depending onthe case definitions, the region and possibly the year(s) underinvestigation [138,431e434]. Coinfections with all three agentshave been reported [433].

In experimental animals, simultaneous infection with Bb andA. phagocytophilum can increase the pathogen load in various tis-sues, markedly alter the cytokine pattern, result in a redistributionof splenic T cell subsets, and enhance the severity of LA, with theprecise nature and combination of effects depending on the mousestrain and the route of infection (needle inoculation versus tick-mediated infection) [435,436]. When tick-transmittedA. phagocytophilum infection preceded tick-mediated Bb infectionby 8 days, the burden of spirochetes, but not of A. phagocytophilum,was enhanced in the heart and certain other tissues, but carditisprevalence and severity were not altered [437]. Together, these datasuggest that A. phagocytophilum can have immunosuppressive ef-fects and thereby enhance disease severity. Such an immunosup-pressive effect may also be operative in humans as indicated by thefinding that A. phagocytophilum coinfection in Lyme disease isassociated with a significantly lower number of Borrelia-specific IL-12-secreting, though not IFNg-secreting cells [438].

In human patients, simultaneous infection with Bb and A.phagocytophilum can increase the number of symptoms comparedto Lyme borreliosis alone [431,433], although this is not a consistentfinding [432]. Whether coinfected patients have leucopenia andthrombocytopenia as frequently as patients with HGA alone is alsocontroversial [431,432]. There are data suggesting that these dis-crepancies are at least partly attributable to different case defini-tions [431], but they also may have been a result of identifying mildsubclinical cases when actively searching for coinfections. Similarly,patients coinfected with Bb and B. microtiwere found to experiencemore frequent and diverse symptoms and a more severe and pro-longed disease compared to patients infected with one or the otherpathogen alone [138,433]. While coinfections with Bb andB. miyamotoi have been described in US patients [439], it currentlyremains unknown to what extent this influences the course orseverity of either disease.

7.3. The host immune response in the pathogenesis of Lyme disease

The Borrelia genome does not encode any known toxins;therefore, it seems likely that much of the tissue damage in patientswith Lyme disease is due to the strong host immune response thatBorrelia spp. induces. In order to establish the contribution of theimmune response to the pathogenesis of the various manifesta-tions of Lyme disease it would first be necessary to elucidate whatconstitutes the most appropriate human immune response toBorrelia infection. However, this is difficult because it remains un-clear whether infected patients who remain asymptomatic or onlydevelop EM do so 1) because of a highly efficient immune response,2) because the size of the inoculum was sufficient to induce sero-conversion, but not large enough to cause clinical symptoms, 3) theinfecting strain was of limited pathogenic potential, 4) because ofother as yet unknown host (genetic) characteristics, or 5) somecombination of several or all of these factors. As long as the answersto these questions are lacking, it remains difficult to distinguishbetween beneficial and pathogenic immune responses.

7.3.1. Immune evasionThe Borrelia spirochete has developed a variety of mechanisms

that allow it to adapt to the remarkably different environments itencounters as it cycles through its host reservoirs and tick vectors.One of these is the ability to rapidly alter the expression of surfaceproteins that allows it to transition from the tick midgut into its

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new host while evading the host immune system. Among the mostthoroughly investigated changes are the downregulation of OspAand the upregulation of OspC when the blood meal starts to arrivein the tickmidgut. OspAmediates spirochete attachment to the tickmidgut, while OspC is required in the initial establishment ofinfection in the vertebrate host [440]. This protein can bind theIxodes salivary gland protein Salp15 and thereby prevents recog-nition by antibodies and antibody-mediated killing. In addition,Borrelia spirochetes express a variety of proteins that can bind andneutralize complement, although their importance in vivo largelyremains to be elucidated [440e442]. Inside the new host, expres-sion of OspC is downregulated under the pressure of the host im-mune response [443,444]. Simultaneously, upregulation of otherproteins can be observed, in particular the VlsE protein [443e445].Recombinational switching between an expression locus and 15silent cassettes results in continuous variation in the sequence ofthis protein, and this antigenic variation is another importantmechanism of immune evasion. In addition, Borrelia spirochetesexpress a variety of surface proteins that allow them to bindextracellular matrix proteins such as fibronectin, laminin, decorin,integrins, and glycosaminoglycans [446]. This combined with theability to co-opt host matrix metalloproteinases (MMPs) is likely tobe important for their dissemination through tissue, but binding toextracellular matrix proteins may also provide a means to evadehumoral immunity by hiding in an immune-privileged site.

7.3.2. EM and the early immune responseAfter a tick deposits Borrelia spirochetes in the skin, an influx of

neutrophils is observed at the bite site in mice [447], as is alsoobserved in needle-inoculated mice and rabbits [448,449]. By thetime the EM lesion develops in rabbits, the initial neutrophil infil-trate is replaced by lymphocyteswith scatteredmacrophages [449].This is similar to what is seen in human biopsy samples[181,412,450,451] and may explain why neutrophils are rarelydetected in human EM lesions [450,451]. At least in vitro, neutro-phils and monocytes are not efficient at phagocytosing unopson-ized Bb [452,453], but are able to kill spirochetes through a varietyof mechanisms [454], including neutrophil extracellular traps(NETs), which are web-like structures with antibacterial propertiesthat arise from a specific type of cell death called NETosis [447]. Yet,this process does not appear to be entirely effective. Isolatedmacrophages are capable of phagocytosing and degrading Borreliaspirochetes in the presence or absence of opsonization [452,455].Therefore, they would be expected to play an important role in theearly defense against infection not only by eliminating spirochetesbut also by secreting cytokines that recruit other immune cells.However, in mice infected via tick bite, there appears to be littleclearance of tick-transmitted spirochetes from the site of tickattachment in the first few days, suggesting that in vivo, macro-phages and neutrophils are not effective in eliminating Borrelia[84]. This may be due to the ability of tick salivary components toreduce phagocytosis of human neutrophils and mouse macro-phages [377].

Recognition of Borrelia Osps and other lipoproteins by toll-likereceptors, in particular (TLR)-2 heterodimers with TLR-1 and TLR-6 in conjunction with the CD14 co-receptor, plays a central role inthe activation of the innate immune system [456,457]. However,full activation of the innate inflammatory response seems torequire phagocytosis of Bb followed by activation of the phag-osomal TLRs, TLR-7, -8 and -9, by their respective bacterial nucleicacid ligands [458e460]. There are indications that other patternrecognition receptors, in particular nucleotide-binding oligomeri-zation domain (NOD)2, also play a role [457,461e463]. TLRs areimportant not only in activating the innate immune response, butTLR-dependent recognition of pathogens by dendritic cells (DCs)

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also is a central element in shaping the adaptive immune response[456]. Large numbers of Langerhans cells have been detected notonly at the periphery of the EM lesion, but also in the adjacent non-erythematous skin [451]. DCs were also enriched in blister fluidraised above EM lesions and displayed surfacemarkers of activationand maturation [464]. Both Langerhans cells and dermal DCs havebeen shown to be capable of phagocytosing, processing and pre-senting Borrelia spirochetes in vitro [465,466]. When pulsed withBb, Langerhans cells and splenic DCs can induce helper T cells tostimulate B cell production of Bb-specific antibodies in vitro [467].When adoptively transferred into syngeneic mice, Bb-pulsedsplenic DCs can also protect mice from infection after challengewith Bb, most likely because of their ability to induce earlier helperT cell responses and Bb-specific antibody production.

In addition, DCs have been shown to play a role in the activationof CD8þ T cells. Borrelia spp. are thought to be mainly extracellularpathogens, and electron microscopically, spirochete-like structuresare often observed to be aligned with collagen fibrils in skin[465,468], synovium [469], ligament [470], and heart [243]. This islikely to be due to their expression of Dbps, which bind to decorinexpressed on collagen fibrils. The role of CD8 cells in a variety ofautoimmune diseases has been shown to be critical not only for thepromotion, but also the prevention of disease [471]. Consequently,the role of CD8þ T cells has long been neglected even thoughseveral lines of evidence suggest that they are an importantcomponent of the immune response to Borrelia infection. Trans-mission electronmicroscopy studies revealed that, upon incubationof normal human skin fragments with Bb, dermal DCs containedspirochetes both in phagolysosomes and free in the cytoplasm, andthe same was observed when isolated dermal DCs were incubatedwith Bb [465]. Such cytosplasmic spirochetes could then be pro-cessed and presented by MHC I to CD8 T cells. Bb lacks the ability torecycle membrane lipids and lipoproteins and, therefore, shedsthem in the form of liposome-like blebs. Liposomes prepared fromBb lipids and lipoproteins as a model for native blebs quicklyreached the cytoplasm of a variety of cells via membrane fusion,whereas uptake via phagocytosis was never observed [472].Importantly, human DCs loaded with Bb-liposomes generated Bb-specific CD8þ T cells capable of killing Bb-liposome-treated autol-ogous T cell blasts. This raises the possibility (to date untested) thatsuch Bb-specific CD8þ T cells could kill cells that have taken up Bbblebs and present Bb antigens. Such cells include DCs, and theirdeath could contribute to the ineffectiveness of the early immuneresponse in preventing spirochete dissemination. In vitro, phago-cytosis of Bb induces macrophages to undergo apoptosis [455].Uptake of antigen-containing apoptotic blebs from such macro-phages by DCs could lead to cross-presentation of these antigens.

EM is thought to arise from the multiplication and spreading ofspirochetes in the skin and their induction of both innate andadaptive inflammatory immune responses. Blister fluid raisedabove EM lesions was enriched for monocytes/macrophages andDCs and these showed an activated phenotype [464]. Both CD4þand CD8þ T cells, particularly of the antigen-experienced memoryphenotype, were also enriched, and this agrees with the observa-tion that CD8þ T cells constitute a substantial portion of the infil-trate in EM and ACA lesions [451,473]. There are surprisingly fewinvestigations of the cytokine production in skin, and these showconsiderable interindividual variation in the protein or mRNAexpression of a large variety of cytokines and chemokines, withIFNg, TNFa, IL-1, IL-6 and IL-10 being most consistently detected[181,464,474e476]. There is, however, no basis for treatment withthe newer biologics and indeed there has been very little dissection,unlike in rheumatoid arthritis, to understand the complexity ofcytokine production and interactions [270,477]. Strong expressionof IFNg in EM lesions and high Bb-specific IFNg recall responses

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during convalescencewere found to be associated with the absenceof residual non-specific symptoms after treatment [475,478]. Thissuggests that IFNg plays a role in early spirochete elimination,which would be consistent with its well-known role in macrophageactivation.

7.3.3. Pathogenesis of LNBThere is evidence that Bb can invade the nervous system early

after infection, but even the detection of Bb DNA in CSF is notnecessarily associated with neurological symptoms [220,479].Thus, it appears that the mere presence of Bb spirochetes is notsufficient to cause clinical disease. On the other hand, the rapidresponse to antibiotic therapy suggests that the presence of viablespirochetes is required for symptomatic LNB. The frequency (up to45%) with which Bb can be isolated from the blood of patients withearly Lyme disease in the US suggests that hematogenous spread isa frequent route of dissemination to tissues like the nervous system[101e103]. In Europe, however, spirochetemia is far less frequentlydetected [104e106]. In addition, in European patients with LNB,there often is a topographical association between the site of thetick bite or EM and the neurological manifestations, such as the firstparesis in adults [187] or facial palsy in children (who are morefrequently bitten in the head and neck region) [189]. Such a topo-graphical association has not been observed in American patients[200]. This suggests that European Borrelia spp. might spread viamigration along peripheral nerves.

In vitro, Bb can adhere to, and be internalized by, neural and glialcells, which may interfere with their function and induce cell death[480,481]. However, cytotoxicity is only seen at high multiplicitiesof infection (MOIs) [480], which are unlikely to be reached in thenervous system given how rarely spirochetes can be cultured ordetected via PCR in CSF, or visualized in biopsy or autopsy material.There is indirect evidence that Schwann cells in peripheral nervelesions of Bb-infected rhesus monkeys produce nitric oxide andTNFa [381]. This is consistent with evidence that Bb can also inducea variety of neurotoxic substances in vitro, including nitric oxideand pro-inflammatory cytokines such as IL-6 or TNFa from glialcells [482] and IL-6, IL-8 and CCL2 from human Schwann cell cul-tures and dorsal root ganglia tissue explants obtained from rhesusmonkeys [483]. Dexamethasone not only reduced the production ofthese mediators but also the Bb-induced apoptosis observed insensory neurons in this model, indicating that apoptosis was due tothe Bb-induced inflammatory response.

Histopathology of peripheral nerve biopsy specimens from pa-tients with neurological manifestations of Lyme disease shows aperivascular infiltrate of lymphocytes, plasma cells and histiocytesin association with patchy multifocal axonal degeneration[179,484e486]. A lymphocytic infiltrate has also been observed inthe meninges [365], extending into the white and gray matter in anadvanced case, along with infiltration of dorsal root ganglia andnerve roots [484,487]. It is likely that these infiltrating immunecells also contribute to the production of inflammatory andneurotoxic substances. T-cell lines and clones isolated from the CSFof patients with LNB show a strong polarization towards a type 1cytokine pattern (high production of IFNg and mostly undetectablelevels of IL-4) [488,489]. When CSF mononuclear cells (MNC) fromadult patients with LNB are stimulated with borrelial antigensin vitro, there is a marked upregulation of the number of IFNg-secreting cells [490e494], and this upregulation is significantlygreater compared to peripheral blood, suggesting a strongcompartmentalization of the Borrelia-specific IFNg response[490,494]. However, the number of responder cells shows pro-nounced variation between patients [490e494], as do the con-centrations of IFNg and essentially all other cytokines andchemokines that have been measured in CSF of patients with LNB

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[495e497]. The actual concentrations of IFNg in CSF are remarkablylow [495,497]. Not only have CD8þ T cells been detected in nervebiopsy samples from patients with ACA-associated polyneuropathy[216], but clonal expansion of CD8 T cells has been demonstrated inthe CSF of patients with acute LNB [498]. The expanded clonesproduced TNFa and some IFNg in response to stimulation withBorrelia antigens. While the role of these Borrelia-specific CD8þ Tcells in LNB remains to be established, their detection in tissues thatare targeted soon after Borrelia infection suggests a role in earlyhost defense. On the other hand, peripheral CD8þ and gd T cellswere identified as the predominant sources of Borrelia-specificIFNg production in patients with chronic Lyme borreliosis (mostlyLNB) [499]. In view of the strong CSF compartmentalization of theimmune response, the relevance of these findings is unclear andthe precise role of this T cell subset remains to be elucidated.

A Swedish group of investigators presented data suggesting thatfailure to upregulate IL-4 production after an initial IFNg responsewas associated with a chronic course of LNB [490,492], as were lowCSF concentrations of TNFa and TGFb [491]. However, this is diffi-cult to reconcile with the observation that highly elevated numbersof Borrelia-specific IFNg-secreting T cells can still be detected insome patients after complete recovery [490,494]. The reportedassociations should be interpreted with great caution since theywere based on small numbers of patients andmultiple comparisonswithout statistical correction, and the statistically significant dif-ferences often seemed to be driven by a small number of patientswhose cytokine responses were markedly higher than the mean[490e492].

A highly consistent finding is that CSF of patients with LNBcontains high levels of B-cell chemoattractants and among these,CXCL13 plays a key in the recruitment of B cells [500,501]. B cellsconstituted an average of 16.5% of all CSF cells in patients with LNB,this proportion being significantly higher compared to patientswith viral meningitis or multiple sclerosis [502]. There are datasuggesting that B cells could contribute to the pathogenesis of LNBby producing antibodies that cross-react with neuronal antigens viamolecular mimicry; molecular mimicry has been a classic mecha-nism of autoimmune disease [503]. Both OspA and the flagellarprotein of Bb share antigenic determinants with neuronal tissuecomponents [504,505], and Bb-specific antibodies cross-reactivewith neuronal antigens have been demonstrated in serum of pa-tients with LNB [506,507]. Humoral immune responses to self an-tigens may also develop independently of molecular mimicryduring Borrelia-induced CNS inflammation [508,509]. These aretransient and most likely arise as a result of tissue damage thatunmasks novel epitopes. The rapid response of the manifestationsof LNB to antibiotics in the vast majority of patients suggests thatsuch autoimmune responses do not contribute to the developmentof chronic disease [510]. However, unlike more classical autoim-mune diseases, there is again very little evidence to suggest using Bcell therapy to treat patients with Lyme disease [511,512]. Whetherand to what extent any of these mechanisms play a role in thepathogenesis of LNB remains to be elucidated.

7.3.4. Pathogenesis of Lyme arthritis7.3.4.1. Murine Bb-induced arthritis. Bb spirochetes can readily becultured from the joints of mice needle-inoculated with as few as102 or 103 spirochetes [448,513,514] and quantitative PCR showsthe ankle to be one of the most heavily infected tissues [368,420].Mice without detectable Bb DNA in the joint essentially show nojoint inflammation, suggesting that the presence of spirochetes isrequired for the development of arthritis [368]. The murine jointinfiltrate consists primarily of neutrophils and other leukocytes,while lymphocytes are sparse [515,516]. In mice infected with Bbvia syringe inoculation, T cells are not necessary for the

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development of arthritis [418,517,518], nor does adoptive transfer ofT cells from Bb-infected animals protect recipient SCID mice fromthe development of arthritis [519]. T cells also do not play a role inthe resolution of arthritis [518,520]. They may actually enhancepathology, as evidenced by the observation thatmice lacking both Band T cells have less severe arthritis and carditis than mice lackingonly B cells [521]. The results of T-cell reconstitution experiments inRag1 knockout mice indicated that T cells, more specifically CD4 Tcells, were primarily responsible for increasing the severity ofarthritis [521]. However, this contrast with other data showing thatdepletion of CD4þ T cells resulted in greater arthritis severity,whereas depletion of CD8þ T cells improved it [513]. In murineLyme disease, T cell help is also not absolutely required for pro-duction of protective and disease-resolving antibodies [522e525].Instead, T cell-independent B cell responses are of major impor-tance in host defense [518,526].

Nonetheless, there are data suggesting that Bb-specific T cellcytokine responses may play a role in the pathogenesis of arthritis.Arthritis-susceptible C3H and the more arthritis-resistant BALB/cmice differ in the production of IL-4 and IFNg during antigen-specific recall responses, suggesting that these prototypical type 1and type 2 cytokines may play a role in disease severity or duration,possibly by influencing tissue spirochete burden [514,520,527,528].However, the findings are inconsistent, hence the contributions ofIL-4 or IFNg to spirochete elimination or disease resolution aredifficult to determine. The results of studies with a variety of knock-outmice show that neither IL-4 nor IFNg are absolutely required forthe development of arthritis in mice infected via needle inoculation[529e532].

Due to the dearth of direct comparisons, it is currently unclear towhat extent the cytokine profile of mice with tick-transmittedinfection differs from that seen in syringe-inoculated mice.Antigen-specific recall responses in mice with tick-borne infectionwere assessed earlier postinfection than in most needle inoculationstudies [374,533] and showed a weak Th1 pattern in BALB/c mice[374]. In contrast, restimulation of lymph node cells from needle-inoculated BALB/c mice with Bb lysate resulted in a weak mixedTh1/Th2 response [374]. Since these cells were harvested 9 dayspostinfection, these data are consistent with the observation thatBALB/c mice infected via needle inoculation started to develop Bb-specific IL-4 production in addition to IFNg secretion at some pointbetween d4 and d14 postinfection [528]. In contrast, stimulation oflymph node cells with ConA yielded a strong Th2 pattern in BALB/cmice infected via tick bite [374], as has been observed by others[533]. This Th2 skewing was shown to be due to tick-transmittedfactors [533]. Arthritis-susceptible C3H mice showed even stron-ger Th2 polarization, and treatment with IFNg, IL-2, and TNFa aloneor in combination during the first 10 days after tick placementprotected these mice from Bb infection, the protection being almostcomplete with the combination of all three cytokines [370].Conversely, neutralization of IL-4 or IL-5 before the placement ofinfected ticks resulted in significant increases in spirochete burdenin various tissues [534].

After infection with Bb via syringe inoculation, exogenous IL-10did not attenuate arthritis inwild-typemice [535], but deficiency ofIL-10 in both C3H and B6 mice was associated with more severearthritis despite a marked decrease in the tissue spirochete burden,particularly in the ankle joint [429,535]. This suggests that IL-10 isvital for establishing the right balance between spirochete clear-ance and protection of host tissue from an overly exuberant in-flammatory response. Indeed, there are data suggesting that theeffects of IL-10 deficiency are due to enhanced macrophage pro-duction of pro-inflammatory cytokines [100], and are partlymediated by deficient downregulation not only of IFNg productionin the joint [536], but also of systemic IL-17 synthesis [537]. IL-17

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and several of the cytokines involved in the differentiation andmaintenance of Th17 cells, such as IL-15 and particularly IL-23, alsoplay a central role in the development of arthritis in the Borreliaspp.-vaccination and -infection model [538e543]. In this model, B6mice are vaccinated with B. bissettii and challenged with Bb 21 dlater, with the vaccination step priming Borrelia-specific T cellswhile infection with a heterologous strain circumvents the pro-tective antibody response. Note that in this model, the infiltrate inthe joint contains lymphocytes in addition to neutrophils andmacrophages [544]. In contrast, IL-17 seems to play at best a minorrole in the standardmurine model, the C3Hmouse infected with Bbvia needle inoculation, which is largely T-cell independent [545].

Numerous studies have also documented that B cells, while notrequired for the development of arthritis, are necessary for remis-sion of arthritis [418,517,518,520,521,546]. Whether they are alsosufficient remains somewhat controversial. For example, in theabsence of natural killer T (NKT) cells, arthritis did not resolve inmice with tick-transmitted Bb infection despite increased anti-Bbantibody production [363].

If B cells and T cells are not required for the development ofarthritis in the commonly used murine models, this suggests thatinnate inflammatory responses make a major contribution to Bb-induced arthritis. While the available data do not suggest a sub-stantial role for NK cells, there is evidence that neutrophilscontribute to the pathogenesis of murine LA via their ability toproduce inflammatory cytokines and chemokines, and data fromthe hamster model also implicate macrophages [544].

7.3.4.2. Pathogenesis of human LA. Perivascular accumulation oflymphocytes and macrophages and scattered mast cells in theinterstitium are typical histopathological findings in patients withsevere, mostly chronic LA [266]. Some samples may also showlymphoid follicles with the cellular composition, though not thearchitecture, of germinal centers. Neutrophils are not generallyseen in the synovium in LA, but it has been hypothesized that theyare present in the acute stages, but are soon replaced by lympho-cytes and plasma cells [365]. Support for this theory comes fromthe observation that histopathology in a patient with LA whoresponded to antibiotic therapy showed granulation-like tissue andan infiltrate consisting mainly of neutrophils, whereas a treatment-resistant patient showed the typical histopathology of chronicarthritis [547]. Neutrophils are abundant in synovial fluid, wherethey often represent ~80e90% of cells [248,548], suggesting thatthey contribute to the pathogenesis of LA. They possess the abilityto produce a variety of inflammatory cytokines, such as IL-1b, TNFaand IL-15. Both TNFa and IL-1b are principal mediators of jointdestruction in other forms of inflammatory arthritis due to theireffects on bone resorption, resorption of proteoglycans in cartilage,and their ability to stimulate the release of additional pro-inflammatory cytokines and other inflammatory mediators. Timeto resolution of an arthritis episode was found to be longest inpatients with the highest levels of IL-1b and the lowest levels of itsnatural antagonist, IL-1Ra [549]. In addition, neutrophils can pro-duce chemokines that attract Th1 cells, Th17 cells or both, such asCXCL10, CCL2, and CCL20 [550]. The concentrations of all of thesechemoattractants were found to be high in synovial fluid of patientswith LA [550].

The initial focus in the assessment of the roles of cytokines in thepathogenesis of LA was on Th1 cells. Immunohistochemistryshowed that IFNg was the predominant cytokine in the synovialmembrane, while lesser numbers of cells expressed IL-4, IL-10 andIL-12 [551]. The Borrelia-specific recall response in synovial fluidMNC from patients with LA is also polarized towards a type 1cytokine pattern [551,552], and such a pattern is also seen in T celllines or clones obtained from PBMC or synovial fluid [553,554],

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which sometimes co-produce IFNg and IL-10, but only rarely IL-4[555]. There are data suggesting that the ratio of Th1:Th2secreting cells in synovial fluid correlates with the severity of Lymearthritis [552] and that high synovial fluid IFNg concentrations areassociated with antibiotic-refractory arthritis [556]. However, noassociation between IFNg mRNA expression in synovial fluid MNCor synovial membrane and responder status was detected [551].

As has also been reported from CSF of patients with LNB [498],Borrelia-specific CD8þ T cell lines could be generated from pe-ripheral blood and synovial fluid of five patients with LA [557].Specific HLA class I-restricted cytotoxic activity could only bedemonstrated with samples obtained after the resolution ofarthritis, not during active arthritis. Unlike what is seen in themurinemodel of LA [513], this seems to imply that CD8 Tcells play arole in the resolution of LA. The detection of cytotoxic activity onlyafter disease resolution may explain why others failed to establishBb-specific cytotoxic T cell clones from PBMC or synovial fluid cellsof patients with chronic LA [553], although this failure may havebeen due to methodological issues rather than to the true absenceof such cells [557]. Unlike rheumatoid arthritis, there has not been acomprehensive genetic analysis [558].

There is increasing evidence that not only Th1 cells, but alsoTh17 cells contribute to the pathogenesis of LA not only in thevaccination-infection model of murine Bb-induced arthritis, butalso in human LA. Synovial fluid of patients with LA was shown tocontain IL-17-producing T cells [559] and significantly elevatedlevels of IL-17 compared to controls [550]. In addition, theneutrophil-activating protein A (NapA) of Bb was detectable in sy-novial fluid of patients with LA, this protein stimulated the pro-duction of high levels of IL-17 as well as IFNg from synovial fluid Tcells, and NapA-specific Th17 cells accumulated in synovial fluid,but could not be detected in peripheral blood [550,560]. NapA alsotime and dose-dependently induced the production of IL-23 byneutrophils and monocytes and of other cytokines involved in thedifferentiation and maintenance of Th17 cells in monocytes. On theother hand, a polymorphism in the receptor of IL-23 was associatedwith significantly lower in vitro production of IL-17 by Bb-stimu-lated PBMC, but did not influence the frequency of arthritis or otherchronic manifestations of Lyme disease [561]. Together, these re-sults suggest that synovial rather than peripheral production of IL-17 contributes to Bb-induced joint inflammation.

It has long been held that tissue damage results from the stronginflammatory response to Bb since the spirochete does not expresstoxins. However, it was recently discovered that Bb expresses aprotease with aggrecanase activity [562], and this may enable it tocause direct damage to joint tissue. In addition, Bb is capable ofinducing the expression of variety of host matrix metal-loproteinases (MMPs) as well as the aggrecanase ADAMST-4, butnot ADAMST-5, by human chondrocytes in vitro [563e566].Furthermore, several MMPs as well as ADAMST-4 are elevated inthe synovial fluid of patients with LA. Additional MMPs weredetected mainly in the synovial fluid of patients with persistentsymptoms after antibiotic therapy [564]. There are data suggestingthat thesewere not directly induced by Bb, but that their expressionresulted from the activation of innate inflammatory responses.Altogether, these data suggest that a variety of enzymes capable ofdegrading the matrix component aggrecan play a role in thedegradation of cartilage and subsequent joint damage in human LA.

7.3.4.3. The pathogenesis of antibiotic-refractory LA7.3.4.3.1. The autoimmune mechanism hypothesis. Compared to

patients with antibiotic-responsive LA, patients with antibiotic-refractory arthritis more frequently possess HLA-DRB1*0401 andrelated alleles [279,319,567], which are also associated with theseverity of rheumatoid arthritis. US patients with LA frequently

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develop seroreactivity to OspA near the onset of prolonged episodesof arthritis [279]. The highest risk of non-response to treatment wasobserved in the group of patients who had both the HLA-DR4specificity and elaborated an anti-OspA antibody response [278].The association of antibiotic-refractory arthritis with certain HLAclass II alleles implicates CD4þ T cells in the pathogenesis ofantibiotic-refractory arthritis. The association of these same alleleswith severity of rheumatoid arthritis suggests that an autoimmunemechanism might play a role in perpetuating the inflammatoryresponse, even after the apparent elimination of infection withantibiotics [117,568,569]. Indeed, PBMC or synovial fluid T cells ofpatients with treatment-resistant LA exhibited a significantlyhigher frequency of proliferative and IFNg responses to theimmunodominant epitope of OpsA (OspA164e173) compared to pa-tients with antibiotic-responsive arthritis [570]. Use of an algorithmto predict epitopes presented by DRB1*0401 resulted in the iden-tification of LFA-1 as a candidate autoantigen, and OspA164e173-specific DRB1*0401 þ CD4þ T cells isolated by tetramer technologyrecognized the relevant epitope of LFA-1 [571]. However, its lack ofbinding to DRB1*0101, the other HLA-DR molecule associated withantibiotic-refractory arthritis, makes its relevance doubtful [571].Other candidates include cytokeratin-10 [572] and endothelial cellgrowth factor [573].

This autoimmune hypothesis ignores that LA eventually resolvesin essentially all patients. It is somewhat difficult to comprehendhow an infection-induced autoimmune process spontaneouslydisappears after it was capable of maintaining joint inflammationfor months or years after the apparent eradication of the pathogen.On the other hand, post-streptococcal rheumatic heart disease alsofades and would be considered as a classic autoimmune disease.Additionally, it is well known that patients with Behcet's syndromeundergo spontaneous remission over time and this is likely alsotrue of a number of auto-inflammatory diseases [574,575]. Inaddition, a humoral response to OspA is not commonly seen inEuropean patients with LA [121,576], and the immunodominantepitope identified in Bb OspA does not exist in B. afzelii or B. garinii[577]. This has been proposed as an explanation for the putativeabsence of antibiotic-refractory arthritis in Europe. However, theavailable data clearly show the existence of treatment-resistant LAin European children [249,259,320], and some adult cases have alsobeen reported [550,551,578,579]. Therefore a different pathoge-netic mechanism must be responsible for antibiotic-refractory LA,at least in Europe.

7.3.4.3.2. The spirochete persistence hypothesis. Borrelia spp.establishes persistent infection in a variety of vertebrate hosts, suchas laboratory mice, rats, hamsters, gerbils, guinea pigs, dogs, andnon-human primates, even though these animals mount a strongimmune response that considerably reduces the spirochete burden.Persistent infection has also been observed in untreated humans[244,580], as most clearly demonstrated by the isolation of spiro-chetes from an ACA lesion of more than 10 years' duration [359].The question then arises of whether Borrelia spirochetes can evadenot only the host immune response but also treatment with anti-biotics and thereby account for the persistent symptoms of patientswith treatment-resistant LA. It has been postulated that they coulddo so by hiding in sites inaccessible to both, such as the extracel-lular matrix. Bb expresses various surface proteins that can bind avariety of extracellular matrix proteins such as fibronectin, laminin,decorin, integrins, and glycosaminoglycans. Electron microscopyshows spirochete-like structures to be aligned with collagen fibrilsin various tissues, including synovium [469] and ligament [470].Another possibility is the intracellular seclusion of spirochetes. Ithas been shown that a several different cell types are capable ofinternalizing Bb in vitro and that spirochetes remain viable forseveral weeks of co-culture [481]. However, it currently remains

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unknownwhether and towhat extent suchmechanisms contributeto borrelial immune evasion and potential persistence after anti-biotic treatment.

In patients with antibiotic-refractory arthritis, borrelial DNA canquite often be detected by PCR in synovial fluid or tissue for up to9 months after the beginning of the first course of antibiotic ther-apy [117,268,569,578,581,582]. However, positive PCR results arenot associated with recurrence or duration of arthritis after therapy[569]. In samples obtained �12 months after the start of therapy,PCR results are generally negative [568,569], although longerpersistence has occasionally been reported in synovial fluid [583]and muscle [584]. Since PCR is not 100% sensitive, a negative PCRresult does not prove the absence of spirochetes. On the other hand,PCR cannot distinguish between viable and non-viable organisms.Attempts to detect borrelial mRNA transcripts as a marker ofmetabolic activity failed in three synovial fluid samples that yieldedpositive PCR results for borrelial DNA [569]. This suggests that thisspirochetal DNA originated from non-viable organisms. In contrast,a few investigators have been able to culture Borrelia spp. fromsynovial samples or tendon for up to ~10 months after antibiotictreatment [112,113,470]. In addition, analysis of plasma cells ob-tained via laser-capture microdissection from joint tissue of pa-tients with antibiotic-refractory LA provided evidence of ongoing insitu diversification and affinity maturation of the antibody reper-toire, even though PCR yielded negative results for Bb DNA [585];the authors suggested that this was due to the continued presenceof an autoantigen; yet, persistence of Bb antigens cannot beexcluded.

Similar to what had previously been described in a treated pa-tient [586], Wormser et al. [587] reported the visualization of a fewmostly non-motile spirochetes in culture medium inoculated withsynovial fluid from an untreated patient. These spirochetes couldnot be subcultured and appeared to be enmeshed in some form ofmatrix of unknown composition. This led to the hypothesis thatthey represent non-viable, but morphologically intact, organismssome component of which might be capable of triggering an in-flammatory response upon release into the joint space [587]. Someconfirmation for this hypothesis comes from experimental animals[588]. Through the use of real-time intravital microscopy and Bbstably transformed to express the green fluorescent protein (GFP),large deposits of nonmotile fluorescentmaterial could be visualizedat the interface of ear dermis and cartilage and in patellar enthesesof both sham-treated and doxycycline-treated Myd88-deficientmice (chosen because of their higher pathogen burden), whilemotile spirochetes were detectable only in sham-treated animals[588]. Immunization of naïve mice with patellar homogenatesinduced seroconversion in the recipients and enhanced macro-phage TNFa production in vitro regardless of the antibiotic treat-ment status of the donor. In a separate experiment, the results ofxenodiagnosis and culture of blood, ear skin and urinary bladderssuggested that no viable spirochetes persisted inwild-type B6 micewith tick-transmitted Bb infection, and only 1/21 Myd88-deficientmice was culture-positive after treatment with doxycycline in thedrinking water. However, borrelial DNA could still be detected inear skin, but not tibiotarsal joints, after antibiotic treatment.Nonetheless, confirmation that the patellar homogenate fromantibiotic-treated animals was unable to induce infection wouldhave been desirable. These results strongly suggest that immuno-genic spirochete material persists near cartilage and in enthesesafter antibiotic treatment and could provide a mechanism for thedevelopment of antibiotic-resistant LA if it could be shown thatthese spirochetal remnants persisted for at least several months.

In contrast to these findings, other studies in mice infected withBb via syringe inoculation or tick bite show the persistence not onlyof borrelial DNA in various tissues, but also of a small number of

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non-cultivatable spirochetes [589e591]. Conflicting results havebeen obtained regarding their infectivity, but they do not causepathology. While tissue DNA levels continuously decrease until8 months after completion of antibiotic treatment, they resurge tolevels similar to those in saline-treated animals at 12 months aftertreatment [592]. Despite declining antibody levels, this is notaccompanied by tissue inflammation, suggesting that viable spi-rochetes survive, but remain non-pathologic. Other animal studieshave yielded inconsistent results regarding the persistence ofviable spirochetes after antibiotic treatment [593]. This may partlybe due to the use of different inoculum sizes, failure to consider thepharmacodynamics of the specific model, and varying outcomemeasures. Of note, in syringe-inoculated rhesus macaques treatedwith antibiotics during the disseminated stage of disease, Bb DNAand RNA (as a marker of viability) could be detected in some of thetreated animals and a few spirochetes could be recovered fromxenodiagnostic ticks [594]. A limited number of slow-growing, butmetabolically active, spirochetes could be cultured from some ofthe animals. Similarly, tissues remained not only PCR-positive, butspirochetes could be cultured from some antibiotic-treated dogswith tick-transmitted Bb infection [593,595].

Correspondingly, Borrelia spp. can occasionally be cultured fromsynovial samples or tendon for up to ~10 months after antibiotictreatment [112,113,470], the site of previous EM lesions up to 3months after therapy [285,596,597], and from CSF of patients withLNB up to at least 7 months after treatment [297,597], even if this isnot always associated with recurrent or persistent symptoms [597].While such cases are rare, they nonetheless suggest that the sur-vival of some viable spirochetes after antibiotic treatment couldoccasionally contribute to some of the long-lasting manifestationsof Lyme disease, including antibiotic-refractory LA.

7.3.5. Potential pathogenesis of PLDSEvidence is emerging that the persistence of symptoms after

antibiotic treatment may be associated with immunological ab-normalities in a subset of patients. In particular, compared to pa-tients with a history of Lyme disease without persisting symptomsafter treatment, a significantly higher frequency of anti-neuralantibody reactivity was seen in convenience samples of patientswith PLDS who had participated in two of the antibiotic retreat-ment trials [326,598]. These anti-neural antibodies reacted with alarge variety of neural tissue components, suggesting that they arenot directly involved in the pathogenesis of PLDS. In Slovenianpatients with EM, there was a strong correlation between theserum concentrations of the Th17-associated cytokine IL-23 atpresentation and the subsequent persistence or development ofsymptoms 2 months after antibiotic treatment [599]. Those withpersistent symptoms more frequently had antibodies againstendothelial cell growth factor, a putative autoantigen implicated inthe persistence of arthritis after antibiotic therapy [573], and levelsof IL-23 also correlated with the concentrations of these autoanti-bodies in this subset of patients [599].

8. Conclusions

Lyme disease is an infectious cause of recurrent bouts of arthritisoften part of a multi-system disease that involves cardiac, skin andneurologic manifestations. It typically begins with an erythematousrash but can also include a number of other dermatologic abnor-malities. Approximately 20% of patients experience only dermato-logic features and do not progress further. However, 40e60% ofpatients eventually develop an inflammatory arthropathy. Thedisease is initiated following exposure of a genetically at risk host toa tick that carries a spirochete. There is now extensive research onthe molecular characteristics of these spirochetal infections,

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including their genomic organization. Virtually all confirmed casesof Lyme disease occur in the Northern hemisphere and there isinformation that there is both under as well as over reporting ofLyme disease. The under reporting likely comes from failure toconsider the diagnosis as well as patients who undergo sponta-neous remission. The over reporting comes from an over zealousdiagnosis based on misinterpretation of the serologic findings andinaccuracies of internet information. Further research to identifythe mechanism of action and effector mechanisms will lead toimproved therapies. Finally, physician education is cristical to anaccurate diagnosis.

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