The diagnosis of Lyme borreliosis is one of the most complex infectious disease challenges in 21st-century medicine. Although the disease has been recognised for more than four decades, there remains considerable uncertainty surrounding diagnosis in clinical practice today. This is partly due to the extremely varied clinical presentation of Lyme disease, and partly to the fact that the vast majority of laboratory tests currently used routinely do not detect the pathogen itself, but merely measure the immune system’s response. This is problematic in itself, but it becomes particularly difficult in the case of a bacterium that has developed highly sophisticated immune evasion mechanisms during its evolution.
Immune evasion strategies
The causative agent of Lyme disease, a member of the subgenus Borrelia burgdorferi sensu lato, is a biologically unique microorganism. This bacterium, which belongs to the spirochetes, differs from many classic bacterial pathogens not only in its slow rate of reproduction, but also in its ability to persist in the host for long periods whilst triggering minimal immunological activity. One of Borrelia’s most important survival strategies is antigenic variation. It constantly alters its surface proteins – the so-called Osp proteins – thereby becoming a moving target for the immune system. By the time the body has developed an adequate antibody response to a given antigen, the bacterium may already be expressing new surface structures. This significantly impedes the effective functioning of the immune system and, at the same time, serological diagnostics.
In the early stages of infection, a significant proportion of patients have not yet developed a measurable antibody response. Although erythema migrans may be of clinical diagnostic value, it appears in only about one-third of cases and is often not recognised. In such cases, the sensitivity of conventional laboratory diagnostics is particularly low. Several studies have shown that standard ELISA or blot tests can yield false-negative results in more than half of cases of early Lyme disease. Although the detectability of antibodies increases in later stages, a further problem arises: antibodies can persist in the body for years, or even decades, in the absence of active infection or symptoms. Thus, a positive serological result alone does not prove current infection or ongoing clinical disease.
The most commonly used laboratory tests
Most current international guidelines consider two-step serological diagnosis to be the standard. The first step involves an ELISA or immunofluorescence test, followed by Western blot (immunoblot) confirmation in the event of a positive or equivocal result. Although this system may statistically improve specificity, it has numerous limitations from a clinical perspective. Furthermore, there is growing evidence that Western blot tests alone yield better results than when ELISA tests are added beforehand as a screening test. At the same time, interpreting Western blots is often difficult; there can be significant variations between individual laboratories, and furthermore, the antigenic structure of the different Borrelia species is not entirely identical. In Europe, the situation is particularly complicated by the fact that the infection is not caused by a single Borrelia species, but by several different species and their genetic variants, including Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto. These can elicit different clinical manifestations and different immune responses. Recently, modified serological tests have emerged as a significant area of development; these measure the immune response triggered by previously unknown Borrelia proteins and/or weight the reactions differently. Although the results are improving, they do not represent a major breakthrough, as the most immunogenic proteins of Borrelia were already covered by previous tests.
Some serological tests may even be available as rapid tests or for use by a doctor (point-of-care), but these always represent a compromise compared to blot tests, which accurately measure the reaction to each individual antigen in the laboratory.
Tests based on serology or the immune response have a practical limitation, which is the development or absence of a measurable immune response.
The principle behind indirect tests
One of the biggest problems in diagnostics is therefore the serological approach itself. The essence of indirect diagnostic methods is that they examine the immune system’s reaction rather than the pathogen itself. According to experience and the literature, not only the routinely requested, TB-supported serological tests, but also the tests based on the T-cell immune response that have become widespread in recent times, do not yield satisfactory results. Laboratory procedures based on the immune response assume that the immune system is functioning properly, reacts at the appropriate time, and produces sufficient and specific antibodies. In the case of Borrelia, these conditions are often not met. The bacterium is capable of modulating the immune response, inhibiting the complement system, and reducing immunological recognition. Research suggests that certain Borrelia proteins inhibit the formation of germinal centres (centrum germinativum), directly influencing B-cell activation and the functioning of the cytokine network. As a result, the infection may often remain ‘immunologically silent’, or the body may generate unexpected reactions due to alterations in the immune response.
The diversity and non-specificity of symptoms
The clinical diagnosis of Lyme disease is a particularly complex task, as the symptoms of the infection are extremely varied, fluctuate over time and are often non-specific. Early detection of the disease is complicated by the fact that the symptoms often mimic other infectious, autoimmune, neurological or psychiatric conditions. Clinical diagnosis is therefore not based solely on laboratory results, but requires a combined assessment of a detailed medical history, potential tick exposure, the temporal course of symptoms, and physical examination. It is particularly important to recognise that a significant proportion of patients do not recall a tick bite, so the absence of such a recall does not in itself rule out infection.
The best-known local clinical sign of early Lyme disease is erythema migrans, which is usually a gradually enlarging, often ring-shaped redness developing at the site of the bite. Although it is considered a classic symptom, it appears in only a third of cases and is often seen in an atypical form. It may present as a homogeneous red patch, a bluish-red discolouration, or a multi-focal skin lesion. Observations suggest it develops more frequently in cases of reinfection, and may sometimes be triggered by antibiotic treatment or changes in the immune system. In the early stages, it may be accompanied by flu-like symptoms – fever, elevated temperature, headache, muscle pain, arthralgia, and marked fatigue – which can easily be misdiagnosed as a viral infection or exhaustion. Many patients already report difficulties concentrating, sleep disturbances or unusual neurocognitive symptoms at this stage.
As the infection spreads, Borrelia can colonise various organ systems, resulting in an extremely heterogeneous clinical picture. In cases of neurological involvement, the leading symptoms include facial paralysis, radicular pain, meningitis, peripheral neuropathy, sensory disturbances or chronic headaches. Patients often complain of memory impairment, ‘brain fog’, concentration problems or psychomotor retardation. In more severe cases, encephalopathy, sleep-wake cycle disturbances, mood lability or even psychiatric symptoms – anxiety, depression, panic symptoms – may also occur. These symptoms are often interpreted as psychosomatic or primary psychiatric conditions, which can further delay diagnosis.
Musculoskeletal manifestations may also be characteristic. Lyme arthritis is usually intermittent in nature, most commonly affecting the large joints – particularly the knees – but migratory joint pain and muscle pain are also common. Some patients report chronic inflammatory symptoms, which occur with fluctuating intensity. In clinical diagnosis, it is important to distinguish these symptoms from other rheumatological conditions, such as rheumatoid arthritis, fibromyalgia or spondyloarthropathies. In certain cases, the immunological activation resulting from the infection may also trigger autoimmune mechanisms, which can further complicate the clinical picture.
In cases of cardiac involvement, Lyme carditis may develop, which can be associated with conduction disturbances, atrioventricular block, palpitations, chest pain or arrhythmias. Although a rarer manifestation, it can represent a potentially serious condition. Some patients experience reduced exercise tolerance, orthostatic symptoms or autonomic nervous system symptoms, which may indicate a disturbance in autonomic regulation. These symptoms are often not initially associated with an infectious aetiology; therefore, Lyme disease may still be an important consideration in the differential diagnosis in such cases.
Establishing a clinical diagnosis is further complicated by the limited sensitivity and specificity of laboratory tests. Serological tests may yield false-negative results in the early stages of infection, whilst they may remain persistently positive following a previous infection even in the absence of active disease. For this reason, the assessment of clinical symptoms and medical history is often of greater significance than a single laboratory result on its own. During the diagnostic process, consultation with a neurologist, rheumatologist, infectious disease specialist or immunologist is often required.
One of the greatest clinical challenges of Lyme disease is that symptoms may change over time, improve spontaneously or recur, meaning the disease can have a fluctuating course. Many patients visit various specialist clinics for years before the possibility of a Borrelia infection is even considered. It is not uncommon for them to be treated with a diagnosis of chronic fatigue syndrome, fibromyalgia, depression, an autoimmune disease or an idiopathic neurological condition. For this reason, an interdisciplinary approach, a comprehensive assessment of symptoms, and the recognition that the infection may underlie a wide variety of seemingly unrelated complaints are of paramount importance in the clinical diagnosis of Lyme disease.
PCR or new genetic methods
Interest in direct diagnostic methods has therefore increased worldwide in recent years. Although PCR tests are, in theory, direct diagnostic procedures, their practical applicability is limited. The sensitivity of PCR is low in samples taken from peripheral blood. One reason for this is the genetic variability of Borrelia; another is that the human genome is present in the blood in quantities that are orders of magnitude greater. It is therefore difficult to find a gene sequence that matches every possible Borrelia gene variant exactly, yet does not bind – erroneously – to human sequences, which are present in much greater quantities, with minor or major errors. Better results can be achieved with cerebrospinal fluid or synovial samples, although a negative PCR result does not rule out infection in these cases either.
Frontline researchers have high hopes for whole-genome sequencing; some methods are already available, albeit at a high cost. These methods more or less successfully amplify and assemble all the genetic code present in the samples, then compare them to existing databases. If, in future, the databases come to contain as many gene variations as possible, the pathogen can theoretically be identified based on the degree of similarity to them.
Genetic methods appear to be on the verge of being suitable for the specific confirmation of Borrelia presence, but they cannot provide a quantitative, diagnostic answer – the presence of the pathogen’s genome does not equate to active infection, nor does it equate to clinical status.
Traditional direct tests and their further developments
Culture, a method frequently used in cases of infection, cannot be applied to pathogens that grow slowly and are sensitive to artificial conditions. Several procedures have been published as experimental methods; however, these require a high degree of precision, and the culture time of several months renders them unsuitable for routine diagnostics. Specific identification following culture is not always straightforward; dark-field microscopy, PCR, or immunofluorescence are generally used.
Microscopy in the past and present
Although Willy Burgdorfer originally identified the pathogen using dark-field microscopy, the method was relegated to the shelf of scientific techniques due to the wide variety of artefacts produced and the tendency to misidentify them as Borrelia. Its use in daily diagnostics has fallen out of favour, and publications only recommend it in cases requiring a high level of expertise.
One of the most exciting developments in the revival of this method is direct microscopic pathogen detection supported by artificial intelligence. Of particular significance in this field is the work of Dr Béla Pál Bózsik and the Hungarian-developed DualDur system. The method represents a fundamental shift in approach: rather than analysing the immune response, it directly detects the presence of live Borrelia in a blood sample.
The DualDur technology employs specialised sample processing (concentration), automated dark-field microscopy and image processing supported by artificial intelligence. The aim of the system is to directly identify spirochetes present in the blood sample and, by performing statistical calculations on them, to provide clinically relevant, validated results.
The advantages of direct methods
One of the most important potential advantages of direct detection methods (PCR, culture, microscopy) lies in early diagnosis. As it is not necessary to wait for the development of antibody production, in theory they may be capable of detecting the pathogen even in the early stages of infection. Furthermore, they may open up new possibilities in seronegative cases, particularly in patients where the clinical picture strongly suggests Lyme disease but conventional serological tests are negative or inconclusive. A further advantage of direct detection technology is that it can be used even after treatment to assess treatment efficacy.
The significance of direct diagnostics is not merely a technological issue, but also reflects a shift in perspective. In infectious diseases, the direct identification of the pathogen has traditionally been the basis of diagnosis. In the case of Lyme disease, however, indirect methods have predominated for decades. The future is expected to move towards a combination of direct and indirect diagnostics, in which the clinical picture, serology, molecular tests and direct microscopic methods complement one another to aid a more accurate diagnosis.
Questionnaires
Based on the experience of major Lyme research centres and doctors, numerous questionnaires focusing on symptoms have been compiled. Some of these are publicly available, whilst others are intended for the doctor’s own use. This ‘secrecy’ is not the result of scientific jealousy, but has arisen because the questionnaires can only provide guidance; therefore, making them public could lead to incorrect self-diagnosis. The diversity and non-specificity of the symptoms, combined with the lack of numerical validation of the questionnaires, could lead even a generally well-trained doctor to draw the wrong conclusion and identify the patient as having Lyme disease.
Doctors generally share the questionnaires on request, and they are even available in publications. To date, there is only one published numerically validated questionnaire, but its key is not available. The so-called Horowitz questionnaire is available in numerous versions, and more recently with the aid of artificial intelligence, but even this does not provide a clearly validated diagnosis. Moreover, in the case of Lyme disease, it is just as possible to have one or a few prominent leading symptoms as it is to have more than ten mild and less specific symptoms. The questionnaire developed and used by the medical team at the Lyme Borreliosis Foundation can be requested free of charge by medical colleagues via the contact details provided. This questionnaire, or variations thereof, is used by numerous Lyme specialists across Europe.
Summary
The diagnosis of Lyme disease therefore remains a significant challenge. Due to the multifaceted clinical presentation of the disease, Borrelia’s immune evasion mechanisms, antigenic variation and the limitations of serological tests, there is still no perfect diagnostic method available today. At the same time, advances in direct diagnostic technologies may open up new perspectives for identifying the actual presence of the infection. Questionnaires can assist in identifying symptoms, but are not suitable for establishing a diagnosis.
Sources:
(C) Lyme Borreliosis Foundation
