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More than 30,000 confirmed or probable cases of Lyme disease occur annually in the United States, though the Centers for Disease Control and Prevention estimate that approximately 300,000 cases of presumed Lyme disease go unreported each year. While Lyme disease often presents with a classic erythema migrans rash, some patients present with non-specific symptoms making the diagnosis of Lyme disease largely reliant on laboratory testing. This Hot Topic discusses scenarios in which testing for Lyme disease is indicated and reviews both recommended and inappropriate testing methodologies. The overall focus is on performance of the CDC endorsed two-tiered serologic testing algorithm and the latest CDC recommendations.

Presenter

Elli Theel, Ph.D., Director of the Infectious Disease Serology Laboratory at Mayo Clinic in Rochester, Minnesota.

Transcript

Our speaker for this program is Dr. Elli Theel, Director of the Infectious Disease Serology Laboratory at Mayo Clinic, Rochester, Minnesota. Dr. Theel will review the epidemiology of Lyme disease and discusses the strengths and weaknesses of available diagnostic testing with a focus on the two-tiered serologic testing algorithm as recommended by the Centers for Disease Control and Prevention.Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.

Dr. Theel, thank you for presenting today.

Thank you for the introduction and to those of you joining us today.

Disclosure Slide

Before we begin, I should note that I have no relevant disclosures.

Utilization Message

And the utilization message for today is that as you view this presentation consider the following important points regarding testing for Lyme disease. How is this testing going to be used in your practice? When should tests be used? And how will testing impact patient management?

Objectives

This presentation is the first in a 2-part series on Lyme disease. The second presentation will be given by Dr. Bobbi Pritt, who will provide information on the newest species of Borrelia associated with Lyme disease, named Borrelia mayonii, which was identified here at Mayo Clinic.

The goals for today’s presentation will be to describe the current epidemiology of Lyme disease and how this infection is transmitted. I will then briefly summarize the common symptoms associated with Lyme disease and spend some time discussing the recommended diagnostic methodologies with a focus on serologic testing and test utilization.

Finally, I will conclude with some brief comments on treatment and prevention strategies.

Borrelia Species Associated with Lyme Disease

So starting with a brief historical perspective, Lyme disease as a clinical syndrome was first described by Dr. Alan Steere in a group of children in Lyme, Connecticut who were originally presumed to have juvenile rheumatoid arthritis.

It wasn’t until the early 1980s that the etiologic agent and tick transmission vector for Lyme disease were identified by the entomologist Dr. Wilhelm Burgdorfer after whom Borrelia burgdorferi was ultimately named.

Borrelia species are mobile, cork-screw shaped spirochetes as you can see in the top  image, and members of this genus can be divided into those causing relapsing fever and those that cause Lyme disease, which will be the focus today.

The species associated with Lyme disease are all members of the Borrelia burgdorferi sensu lato complex of which there are currently more than 15 species. Of these, 3 are associated with the majority of Lyme disease cases worldwide as shown in the table below. In North America, until recently, Borrelia burgdorferi  sensu stricto was the only causative agent of Lyme disease and as mentioned earlier, Dr. Pritt will discuss the newest agent, B mayonii, in a subsequent Hot Topic. In Europe and Asia, the primary species associated with disease are B garinii and B afzelii, though B burgdorferi is also encounter in these regions. All of the Lyme disease Borrelia species are transmitted by Ixodes species ticks, and in the United States, this is primarily I. scapularis.

Transmission of Lyme Disease

So how are Borrelia species and Lyme disease transmitted?

Well, this all revolves around the tick lifecycle. Starting with the tick eggs, these will hatch and immediately molt into larva, which will seek out a small rodent, typically a field mouse to feed on. Mice are a natural reservoir for Lyme disease Borrelia in the environment and are the source of larval exposure to B burgdorferi. Larva subsequently molt into the nymph stage, which will feed on a second host, which is typically a small mammal, though nymphs are also the most likely stage to feed on unsuspecting humans. Studies have shown that a B burgdorferi carrying tick has to be attached for at least 36 hours in order for the bacteria to be efficiently transmitted to the individual. Importantly through, humans are considered incidental or dead-end hosts for Borrelia species as we cannot transmit the bacteria back to a naïve tick.

Following the second feeding, nymphs will molt into adult ticks, which prefer to feed on larger animals such as deer, after which females will lay eggs, completing the lifecycle, which in total takes approximately 2 years.

Lyme Disease Epidemiology

As many of you know, Lyme disease continues to be the most common tick-borne infection both in the United States and in Europe, and the number of reported cases continue to increase each year. For example, in 2001, there were approximately 17,000 cases of Lyme disease reported to the CDC and you can see the origin of these cases here, with the 2 major endemic areas in the upper midwest and the coastal northeast states.

Twelve years later, the geographic distribution of Lyme disease cases has significantly expanded and the number of reported cases have more than doubled to over 36,000. However, due to the imperfect nature of surveillance systems, the CDC actually estimates that anywhere from 288,000 to 329,000 cases of Lyme disease occur annually in the US. And perhaps what is more staggering is that 96% of these cases still only occur in 15 states.

It is estimated that over 65,000 Lyme disease cases occur annually in Europe, and with respect to testing, in 2014, approximately 2.4 million specimens were submitted for Lyme disease testing in the United States at a cost of roughly $492 million.

Common Clinical Manifestations of Lyme Disease

The clinical manifestations of Lyme disease can be categorized into 3 general stages, though these features can overlap between stages.

Following transmission, the incubation period prior to development of symptoms is typically 7 to 14 days, though this can range anywhere from 3 to 32 days. The first phase of infection is referred to as early localized disease, which can last from days to a few months post tick-bite. The most common manifestation of Lyme disease is the expanding erythema migrans rash or "bulls-eye-rash," which occurs in up to approximately 80% of individuals. Some examples of EM rashes are shown below. Additionally, this stage is may also be accompanied by nonspecific symptoms, including fatigue, malaise, myalgia, headache, and lymphadenopathy.

Progression to the second stage of Lyme disease, or the early disseminated stage, occurs primarily in untreated individuals anywhere from weeks to months following initial tick bite. Complications that can occur during this phase include development of neurologic disease, which classically presents with the triad of meningitis, cranial neuropathy, and motor or sensory radiculoneuropathy. The facial nerve is most commonly affected, often leading to Bell’s Palsy. Musculoskeletal manifestations, more specifically monoarticular or oligoarticular arthritis, may also develop at this stage and can be either intermittent or persistent and typically affect the large joints. Finally, multiple EM lesions may develop at this stage, as can, more rarely, carditis.

Late Lyme disease is defined as occurring months to years following the original tick bite and manifestations are largely similar to those of early disseminated Lyme disease, including musculoskeletal and neurologic manifestations.

Importantly, the 3 main Borrelia species associated with Lyme disease are also associated with certain clinical manifestations. For example, B burgdorferi is most often associated with development of arthritic symptoms, while in contrast, B garinii is considered to be the most neurotropic of these species, while B afzelii infection leads to significant skin manifestations, including the late manifestation of acrodermatitis chronica atrophicans.

Lyme Disease Laboratory Testing

When it comes to laboratory testing for Lyme disease, it is important to understand when diagnostic testing is most effective.

In the case of Lyme disease, if the patient has a classic erythema migrans rash alongside appropriate exposure history, testing is not recommended as the presence of that lesion is diagnostic. As we will discuss, serologic testing at this stage has a low sensitivity and, therefore, may possibly lead to a false-negative result. Additionally, testing is not recommended in patients who lack symptoms associated with Lyme disease and in those who lack an exposure history.

Finally, there are a number of assays that the CDC and other health agencies do not recommend due to lacking clinical validity, and these include urine antigen tests for Lyme disease, lymphocyte transformation tests, and identification of cell wall deficient B burgdorferi forms.

Laboratory Testing Methodologies for Diagnosis of Lyme Disease

Lyme disease may be diagnosed by a number of methods, though the most common of these and the one we will focus on today is serology.

Briefly, while Borrelia spirochetes may be observed on histopathology from tissue biopsies collected at the expanding EM lesion, histopathologic examination is rarely necessary due to the diagnostic value of the pathognomonic EM rash itself.

Culture for Lyme disease Borrelia species is possible and can provide a definitive diagnosis. However, there are a number of limitations associated with culture, including its variable and low sensitivity and a fairly slow turnaround time, which limits its utility in the acute setting. For both of these reasons, culture is not routinely recommended and, in fact, is no longer routinely available through hospital or reference laboratories.

The 2 methodologies we will focus in a bit more detail will be Nucleic Acid Amplification Tests or NAATs, and Lyme disease serology.

Lyme Disease Laboratory Diagnosis: Molecular

The molecular assay offered through Mayo Medical Laboratories has been validated on a number of sources, including synovial fluid, CSF, blood, and tissue. This assay is advantageous in its quick turnaround time and high specificity. The assay was designed with the goal to differentiate infection with B burgdorferi from the 2 European strains, B garinii and B afzelii. Also, as you’ll hear about in the second part of this Hot Topic series, this was the assay that detected the newest member of the B burgdorferi complex, B mayonii.

Despite these advantages, a number of limitations exist. First, depending on the specimen source, sensitivity can be low. For example, while the NAAT sensitivity for B burgdorferi is approximately 65% from EM tissue biopsies, it is approximately 10% from blood for all species except B mayonii. It is also important to note that almost all individual who are positive by NAAT testing, regardless of the source, are also positive by serology. Many individuals with Lyme disease who are serology positive, however, are not NAAT positive and, therefore, a negative molecular test for Lyme disease does not rule-out infection.

Lyme Disease Laboratory Testing

Due to the limitations of the methodologies we just discussed, serology remains the recommended testing method by the CDC for diagnosis of Lyme disease. More specifically, the CDC endorses the 2-tiered testing algorithm, which we at Mayo Clinic and Mayo Medical Laboratories continue to follow.

So how does this algorithm work? First, all serum samples are initially screened for the presence of total antibodies to Borrelia, most often by an enzyme immunoassay or EIA. If the screen is negative, this may indicate one of two things. First the patient may not be infected with Borrelia and providers are encouraged to consider an alternate diagnosis. Alternatively, the sample may have been drawn too early following transmission for antibodies to be detected. For these individuals, who also meet the criteria of exposure to an endemic area and who present with symptoms consistent with early lyme, a convalescent sample should be submitted in 2 to 3 weeks.

If the initial screen in positive or equivocal, supplemental testing by an immunoblot specific for IgM and IgG class antibodies is required. For patients who have had symptoms for less than 30 days, both the IgM and IgG results should be considered. For patients with more than 30 days of symptoms, only the IgG should be considered as the IgM result, the less specific of the 2 blots, may be present due to a previous infection.

Finally, this algorithm is specific for use in serum only. For diagnosis of Lyme CNS infection or neuroborreliosis, a Lyme Antibody Index assay is recommended, which compares the level of antibodies in normalized serum and CSF. Discussion of this particular assay is beyond the scope of this Hot Topic.

Review of Assays for 2-Tiered Testing

So focusing in on each of these tiers, there are multiple screening ELISAs available commercially for use as a first-tier test. These ELISAs primarily differ in their antigenic composition. Some ELISAs use a whole cell Borrelia burgdorferi sonicate as the adhered target antigens, whereas other ELISAs use unique, recombinant, immunodominant surface antigens for antibody detection.

Due to the conserved nature of these surface-expressed proteins, these ELISAs are capable of detecting antibodies to all members of the Borrelia burgdorferi complex. However, their specificity is imperfect and false-positive results can occur in both healthy individuals living in nonendemic areas and in individuals with other infectious or noninfectious conditions. For this reason, the CDC continues to recommend supplemental testing by immunoblot of positive or equivocal first-tier screening ELISAs.

Moving on to second tier testing, evaluation of both IgM and IgG antibodies individually by immunoblots is recommended, depending on stage of infection. As mentioned earlier, IgM antibodies can remain elevated and detectable for months following infection, therefore in individuals with more than 30 days of symptoms, IgM-positive results are of limited value.

Immunoblots used in the United States and in Europe differ both in the antigens used and the applied interpretive criteria as I will show you on the next slide. Important to remember is that blots used in the United States are specific for infection with Borrelia burgdorferi and a number of studies have shown that their sensitivity for detection of antibodies to the European strains is low–ranging from 10% to 70%. Therefore, individuals infected in Europe may be negative by the assays available in the United States and directed testing using European-based Borrelia blots is recommended.

Lyme Disease Immunoblots

So these are examples of the US and European versions of the respective Lyme disease IgM and IgG immunoblots. Starting with the US version, there are 3 antigens on the IgM and 10 antigens on the IgG blot. For a blot to be considered positive in the United States, the CDC requires reactivity at at least 2 out of 3 bands for the IgM, and 5 out of 10 bands on the IgG blot.

For the European blots, while there are some minor differences between manufacturers and countries, the interpretive criteria are fairly similar. In this particular set, there are 5 antigens on the IgM strip and 11 antigens on the IgG strip. For blots to be considered positive, the requirement is reactivity of at least 1 out of 5 bands on the IgM and at least 2 out of 11 bands on the IgG. So as you can see, the criteria are significantly less strict for interpretation of the European blots; however, the antigens used also differ from those on the US blots.

General Performance Characteristics of the 2-Tiered Testing Algorithm

So a question that comes up often is how well do these assays perform for diagnosis of Lyme disease. Focusing on just performance in the United States, this table shows the sensitivity of the 2-tiered testing algorithm using 2 different screening ELISAs, either a whole cell sonicate or the C6 recombinant peptide ELISA following by immunoblot. And what you can see is that in early localized disease, sensitivity is poor, only about 35% of individuals with EM or other signs of early Lyme disease will be positive by serology. For this reason, patients with EM should not be tested and in all other cases, convalescent testing is required as documentation of IgM or IgG seroconversion is definitive evidence of infection.

Importantly however, the specificity of the 2-tiered testing algorithm, regardless of what screening ELISA is used, is high, over 99% in both healthy individuals and those with other infectious or noninfectious conditions.

As mentioned earlier, because we use B burgdorferi specific immunoblots in the United States, infection with other Borrelia species is unlikely to be detected by the current algorithm and specific testing is required.

Finally, we do not have time to discuss this today, however, a significant amount of work has shown that modification of the current ELISA/blot algorithm to a dual ELISA algorithm improves sensitivity at all stages of Lyme disease, without affecting specificity. Studies are ongoing to further evaluate this new strategy.

Treatment and Prevention of Lyme Disease

To conclude, effective antimicrobial treatment regimens for Lyme disease are available and have been evaluated in clinical trials. Oral antibiotics such as doxycycline or amoxicillin are often prescribed for the common symptoms of Lyme disease, while intravenous antibiotics such as ceftriaxone are reserved for more complicated disease manifestations including neuroborreliosis.

While detection of Lyme disease is important, Ixodes ticks can also transmit other infectious agents, including Babesia microti and Anaplasma phagocytophilum, alongside Borrelia burgdorferi. Clinicians are therefore encouraged to consider ruling out coinfection with these agents as well.

Finally, prevention of Lyme disease is largely dependent on avoidance of tick bites. This involves use of DEET or permethrin-treated clothing when enjoying the outdoors and wearing long sleeved clothes.

While a Lyme vaccine was commercially available in the early 2000, it has since been pulled from the market due to unforeseen side effects. New Lyme disease vaccines are currently in clinical trials and will hopefully be an added tool against Lyme disease in the future.