Lyme disease, or Lyme borreliosis, has become the most common tick-borne disease in parts of the North-Eastern USA and Europe. Lyme borreliosis is caused by spirochetes of the Borrelia burgdorferi sensu lato group. In the USA, Borrelia burgdorferi sensu stricto, from here on referred to as B. burgdorferi, is the causative agent, whereas in Europe also B. burgdorferi, but mostly Borrelia garinii and Borrelia afzelii are the predominant causative agents. In 2009, Dutch general practitioners were estimated to have diagnosed Lyme Borreliosis 22.000 times. This number has increased from 6.500 in 1994, 13.000 in 2001 and 17.000 in 2005.
The disease is named after the town Old Lyme, CT, USA where Prof. Dr. Allen Steere in 1975 first discovered the link between a tick-borne disease and a group of children suspected of juvenile arthritis. Seven years later, the causative agent B. burgdorferi was discovered by Willy Burgdorfer. In Europe, syndromes already reported since 1883, such as Bannwarth syndrome, can retrospectively be seen as Lyme manifestations. Also, the link between tick bites and these manifestations was discovered by Garin and Bujadoux, and it was known that these disorders were caused by a pathogen sensitive to antibiotics. A further insight of the history of Lyme disease in Europe can be found in an elegant review from Stanek and Strle.
The pathogen
B. burgdorferi belongs to the order Spirochaetales, known for its helical shape and motility derived from the flagella. The genome comprises a linear chromosome and 21 linear and circular plasmids, together roughly 1.5x106 base pairs. The genome contains 1780 genes of which none are yet known to encode for toxins, and a large portion encodes for lipoproteins. The obligate enzootic life cycle of the spirochetes involves ticks and a variety of vertebrate hosts, including small rodents, large mammals and birds.
The vector
B. burgdorferi is transmitted by the deer tick, Ixodes scapularis, whereas the European species are transmitted by the sheep tick, Ixodes ricinus, also vector of the tick-borne encephalitis virus. In general, uninfected tick larvae acquire the bacterium by feeding on infected animals. Ticks remain infected during their consecutive molting periods, enabling both nymphal and adult ticks to transmit spirochetes to other (larger) animals, including humans. After their final bloodmeal adult female ticks, which have already mated, lay uninfected eggs; vertical transmission only rarely occurs. Both Ixodes species are capable of transmitting spirochetes while feeding, which can take up to 5 days. The number of visits to the GP's office for tick bites in the Netherlands raised from 371 per 100.000 in 2001, to 446 and 564 in 2005 and 2009 respectively. 18,6% of adult ticks are infected, compared to 10,1% in nymphs, showed by a European meta-analysis in 2005. In 2007, 38% of all tick bites in the Netherlands happened in forests, 36% in gardens and 10% in dunes.
Pathogenesis
A striking feature of Borrelia is its diverse capability to evade the host immune response. Borrelia has several mechanisms for this, but essential is the expression of outer surface proteins (Osp's). The differential expression of Osp's increase the chance of survival in the different environments that the bacteria encounter during their enzootic life cycles. OspA and B are upregulated when migrating from host to feeding ticks, and increase the survival of B. burgdorferi in ticks. OspC is essential for transmission of Borrelia during the tick feeding to the host.
Another evasions mechanism is the recombinant gene expression of the variable major protein-like sequence (vls) locus. This results in altered antigenicity of the lipoprotein VlsE and thus protection against anti-VlsE antibodies. Also, complement resistant Borrelia strains express complement regulator-acquiring surface proteins (CRASPs) enabling Borrelia to bind the host complement system's negative regulators factor H and/or factor H like protein 1. Recently, another protein suggested to be important for evasion of the host adaptive immune response during infection is Lmp1. It consist of a N, middle and C region. The middle region contains proteins associated with adhesion, the C region contains proteins associated with protein-protein interactions. Utpal et al. showed that the N-region increase pathogen survival pathogen survival in-vitro and in-vivo. (51)
The spirochete also exploits tick gut and salivary proteins (salp's), to protect itself. In the tick gut, OspA binds the tick receptor of OspA (TROSPA) and when transmitted from tick to host, OspC binds Salp15. Immunisation studies showed the benefit of these interaction for B. burgdorferi. The OspC-Salp15 binding shields the spirochete from borreliacidal antibodies in Borrelia burgdorferi-immune mice. Furthermore, we showed Salp15 exerts immunosuppressive activity in vitro; inhibiting murine T-cell activation and suppressing human dendritic cell (DC) function.
A number of adhesins, proteins on the outer membrane that are involved in the anchoring and interaction with host cells, have been discovered, and are important for the establishment of infection. A review from Coburn et al overviewed these adhesins, most important are the binding of BBK32 to fibronectin, P66 and BBB07 binding integrin and DbpA and DbpB binds decorin and glycosminoglycans.
Clinical
Early Lyme Borreliosis (days to weeks)
Ticks attached shorter than 24 hours are estimated not to be able to transmit Borrelia. In a Dutch study with 167 tickbite cases in a general practinoners populations, only one case (0,7%) developed Lyme disease, which was after a tickbite longer than 24 hours. Erythematous skin lesions smaller than 5 cm starting within 2 days after detachment of the tick are most likely a tick bit hypersensitity reactions. Tick bite hypersensitivity should disappear within 1-2 days. No diagnostics or treatment after a possible tickbite without the clinical diagnosis of erythema migrans (EM) or Borrelial Lympocythoma, the symptoms of early Lyme borreliosis are recommended. A typical Erythema migrans is an expanding erythematous skin lesion with central clearing located at the site of tickbite starting after 3-30 days, variating from 5 to 75 cm. Symptoms that can accompany are fever, myalgias and arthralgias and locally itching, burning, and mild pain. A Borrelial lymphocytoma is diagnosed seldomly, and only in Europe, but described as a bluish red tumor-like skin infiltrate, often located at earlobe or nipple and more common in children with a subacute onset and can spontaneously resolve. Both these symptoms respond well to antibiotic therapy. In a Swedish study in 1995, 77% of all Lyme manifestations were Erythema migrans and 3% Borrelial lympocytoma.
Early disseminated Lyme Borreliosis (weeks to months)
When the infection is untreated, the spirochete can disseminate and cause early neuroborreliosis (16% of Lyme manifestations), Lyme arthritis (7%), and seldom a (myo)carditits with (partial) atrioventricular block(<1%). Notably, since the late 1980s, increasing awareness for erythema migrans and better effective antibiotic regimes made these clinical manifestations become less common. The European Union Concerted Action on Lyme Borreliosis (EUCALB) has made criteria for these manifestations for clinical purposes and these are also used in the Dutch guideline developed by the CBO. Early neuroborreliosis can be presented with one or more of the following; lymphocytic meningitis, which is more common in the United State, cranial neuritis (Bell's palsy) or a painful radiculitis, which is more common in Europe. Most cases occur between July and November, due to the lifecycle of the tick. In most patients, acute neurological symptoms improve or resolve in several weeks to months, even without antibiotic treatment.
The infection in joints is oligoarticular, 50% occurs in the knee. This manifestation is mostly observed in the United States, where 60% of untreated patients develop arthritis. Cardiac involvement in early Lyme borreliosis is rare, symptoms are related to atrioventricular conduction abnormalities and mostly in the presence of erythema migrans or within a few weeks after onset of infection and seems to be transient and self-limiting.
Late Lyme borreliosis (months to years)
Persistent Lyme borreliosis can present with by joint swelling, skin rash, weakness of facial and other muscles or severe headache. Manifestations can be divided in two groups, one where persistent B. burgdorferi infection is causative for the ongoing symptoms, e.g. acrodermatitis chronica atrophicans (ACA) and neuroborreliosis, and one where other mechanisms play a role, e.g. antibiotic-refractory Lyme arthritis, dilated cardiomyopathy. B. afzelii is associated with ACA, B. garinii with neuroborreliosis and B. burgdorferi with Lyme artritis. 50 to 70 percent of the patients with late manifestations of Lyme borreliosis do not remember an EM
ACA can develop up to ten years after infection and is described as a bluish-red atrophic skin lesion, initially combined with edema and is located on the plantar sites of hands and feet or distal parts of the legs. Periarticular nodules or sclerotic lesions can be observed. ACA can be confused for vascular conditions, more often when the legs are affected. It occurs mostly in women older than 40 years, but has been described incidentally in children.
The late manifestations of neuroborreliosis include encephalomyelitis, encephalopathy and axonal polyneuropathy, for a period of at least 6 months. Symptoms are mainly cognitive, but can also include sensory polyneuropathy or spastic paraparesis, but often presented in the combination of aspecific symptoms, such as fatigue, malaise and myalgia, which makes diagnosing difficult. Pleiocytosis, combined with intrathecal evidence of B. burgdorferi infection are therefore essential. A European study showed that isolated chronic polyneuropathy, without the presence of other late Lyme borreliosis manifestation such as ACA, is rarely caused by Borrelia infection.
Joint manifestations can occur months to years after exposure, with intermittent recurrent attacks occur that persist for days, weeks, or months and are typically asymmetrical and pauciarticular in nature and involve one or two larger joints and almost always the knee. Most Lyme arthritis patients respond well to conventional antibiotic treatment strategies, such as doxycycline, but a small percentage will continue to have chronic joint inflammation, not due to persistence of the spirochete. This is called antibiotic-refractory Lyme arthritis and occurs more often in the United States than in Europe and has been associated with a genetic predisposition, i.e. the presence of several HLA-DR rheumatoid arthritis alleles, among which is the DRB1*0401 molecule.
Dilated cardiomyopathy has been found in patients in Europe based on isolation of the spirochete from heart tissue and serological studies. Since spirochetes have rarely been isolated by culture, this might indicate that symptoms could be due to past infection and myocardial scarring rather than ongoing inflammation due to the presence of the spirochete. Routine therapy and screening of patients with idiopathic dilated cardiomyopathy are of limited utility and should be reserved for patients with a clear history of antecedent Lyme borreliosis symptoms or a tick bite.
It becomes more difficult when patients present with persisting aspecific symptoms after adequately treatment with antibiotics, implying that these symptoms are not caused by persisting B. burgdorferi infection. Therefore, this complex of aspecific symptoms should best be referred to as post-Lyme disease syndrome. Post-Lyme disease syndrome has been linked to a broad array of symptoms that are highly prevalent in the normal population. This, in combination with the fact that specific antibodies against B. burgdorferi occur in approximately 4-8% of the normal population, even up to 20% in high endemic areas, makes the diagnosis 'chronic Lyme borreliosis' a cumbersome diagnosis. A critical appraisal of chronic Lyme Disease has been made by Feder et al. in 2007.
Diagnostics
The diagnosis of Lyme borreliosis is predominantly based on clinical symptoms and Lyme-serological tests. The diagnosis of Lyme borreliosis can be readily considered in case of symptoms which have been associated with B. burgdorferi infection and there is also serological evidence for Borrelia infection (table). Erythema migrans is a clinical diagnosis and serological evidence is not necessary. Furthermore, 20-50% cases of erythema migrans occur without detectable antibodies. On average, antibody response starts 3-6 weeks after infection, but this period has a wide interindividual spreading. In case of an uncertain or atypical EM, serological testing can be considered. Notably, in the absence of specific clinical symptoms, the presence of specific antibodies does not necessarily indicate the presence of an active Borrelia infection. There is no specific test that indicates persisting Borrelia infection. Therefore, a positive antibody test may also be due to clinical or subclinical infections in the past. In the United States in about 10% of the infected individuals asymptomatic infection with B. burgdorferi seems to occur. In Europe this percentage is thought to be much higher. Alternatively, a positive serological test in the absence of specific clinical symptoms could also indicate a false-positive result. Therefore, international and the CBO guideline recommends that, when there is only a small suspicion on Lyme Borreliosis, not to test for antibodies against Borrelia.
Validated and widely accepted diagnostics include culture, Polymerase Chain Reaction (PCR) and antibody serological tests; Enzyme-Linked Immuno Sorbent Assay (ELISA), immunoblot and Western blot. Clearly, a positive culture in the presence of ongoing specific symptoms indicates an active infection and should be considered as the 'gold standard'. Unfortunately, there are limitations to culture. It is expensive, the tissue samples should be incubated in special medium for weeks and there is limited availability in the Netherlands, although it can be performed in certain laboratories, such as the Academic medical Center in Amsterdam (AMC) and Leiden (LUMC). Detection of Borrelia using both culture or PCR can be useful on skin biopsies from patients with EM, acrodermatitis chronica atrophicans (ACA) and in synovial fluid from patients with Lyme artritis, for which a sensitivity of 50-70% has been reported.
Detection of antibodies in serum directed against B. burgdorferi is the most common diagnostic test. Several guidelines recommend that a B. burgdorferi ELISA should be used as a screening test and, when reactive, should be confirmed by an immunoblot or Western blot. ELISAs need to be at least second generation tests. Antigen used should include OspC, but also purified intact flagella can be used. For immunoblot, testing in Europe is different compared to the USA. In Europe, there is a narrower spectrum of Borrelia proteins, compared to the USA. The spectrum of Borrelia proteins recognized on Western blot also depends on the stage of the disease. During early infection, there are only few Borrelia proteins recognized, where as in a stage with ACA a wider spectrum of antibodies can be detected. A European multicenter study has provided a framework for immunoblot interpretation, because a wide range of proteins, i.e. bands, are detected in assays. Eight bands were found suitable for effective discrimination. Newer serological tests include an ELISA detecting antibodies against C6, a 26-amino acid peptide that reproduces the sequence of the sixth invariable region (IR6) within the central domain of the VlsE protein of B. burgdorferi sensu lato. The C6 ELISA has a high specificity but a low sensitivity in the early phase. Notably, antibiotic treatment during early phase of infection cause a decrease in antibody titers against B. burgdorferi. In contrast to antibody levels in early Lyme borreliosis, detection rates of IgG antibodies are nearly 100% in late Lyme borreliosis manifestations. Overall, the specificity of IgG-components in ELISA's varies between 80 and 95%. The specificity of IgM-components is lower, because rheumafactor, acute EBV and CMV infection and MS and other auto-immune diseases can also give a positive test. Although IgG and IgM ELISA's have a high specificity and sensitivity, results were found to be highly variable between different laboratories.
In addition, by some, besides skin biopsies, blood and urine are also used for PCR to detect B. burgdorferi. PCR on body fluids is not recommended for microbiological diagnosis, for one, since Borrelia DNA can be detected in urine from healthy seropositive individuals, making the biological and clinical relevance of a positive urine PCR unclear. In addition, a meta-analysis showed an overall sensitivity of the urine Borrelia PCR of 68% (range 13% to 100%). Finally, a study by Steere and collaborators showed that, after establishing an optimal PCR protocol with spiked urine, in only 1 of 12 patients with an acute infection, presented by an EM, Borrelia DNA was detected.
The presence of anti-B burgdorferi antibodies in the CSF supports the diagnosis of neuroborreliosis. As with serum, also positive B burgdorferi serology in the CSF combined with neurologic symptoms is not always equivalent with neuroborreliosis. A French study showed that from 123 patients with positive IgG burgdorferi serology in the CSF, 74 patients did have another etiologic diagnosis. However, only 2 had a positive intrathecal anti-Borrelia antibody index (AI) (specificity 97%). In the group with definite Lyme, 30 out of 40 had a positive AI (sensitivity 75%).
Therapy
Antibiotics are effective in all manifestations of Lyme Borreliosis, but is most effective in the early stage. The recommended treatment of the different manifestations of Lyme borreliosis are shown in table 2 and has been based on the international guideline of the IDSA and the Dutch CBO guideline. The difference between antibiotics and expectative policy for Erythema migrans has never been studied, because the justified use of antibiotics has been showed by randomized double-masked trials in which different antibiotics were compared. Oral treatment in early Lyme borreliosis is as effective as parental antibiotics, but has lower risks and adverse events.
For EM and Borrelial Lympocytome doxycycline for 10-14 days is effective and without adverse event. The duration of treatment has not been optimalized, but a randomized double-masked trial in 2003 supported that 10 days doxycyline was enough to eradicate Borrelia. For early disseminated Lyme borreliosis doxycycline is also recommended, except for neuroborreliosis for which ceftriaxone is first of choice. This also accounts for late manifestations of Lyme disease, but with a longer duration of treatment.
Several randomized placebo controlled clinical trials did not show beneficial effects of antibiotics over placebo in patients with residual complaints after previous adequate treatment for B. burgdorferi infection. Still, patients with post Lyme disease syndrome who seek medical attention are commonly treated for months to years with (multiple) intravenously administered antibiotics, with a high incidence of serious adverse effects. Occasionally, these patients are treated with unconventional and highly dangerous methods.
Prevention
The best preventive method is avoiding exposure to ticks and reducing the risk by protective clothing and awareness for tick bites.Ticks can be controlled by acaricides, such as cyfluthrin, carbaryl or deltamethrin. However, resistance in ticks occurs, and acaracides are harmful for humans, animals and environment. Calculations have indicated that a vaccine is economically attractive when used for persons with an annual risk of more than 1% of contracting Lyme disease. The only licensed Lyme vaccine was based on OspA, which showed a 70% efficacy in phase III human trials It became available in 1998 but was removed from the market in 2002 because of public perceptions on adverse events. Still, a vaccine based on antibody-antigen from ticks or Borrelia might become an alternative in the future. Animal studies give supportive evidence on immunity against tick. We recently reviewed the possibilities for a vaccine against Lyme borreliosis, such as vaccins based on the combination of Borrelia and tick (saliva) proteins. Antibodies against Salp15, by itself able to impair a B. burgdorferi infection in mice, had a synergistic effect with other antibodies against B. burgdorferi antigens, such as OspA or OspC. This has also been demonstrated for OspA-Dbpa. A triple combination vaccine of OspC, DbpA and BBK32 showed an higher effectivity then single our double, However, this is all in an experimental stage.
