Abstract / Summary
Handling patients with possible Lyme disease and co-infections in clinic and practice, the author reflects on the justifications of his actions as he is constantly drawn into the discussion between theoreticians and practitioners. The representatives of these two opposing camps are organised by the IDSA (Infectious Disease Society of America) and the corresponding European organisations such as EUCALB (European Union Concerted Action on Lyme Borreliosis) on the one hand, and the ILADS (International Lyme and Associated Diseases Society) and the DBG (Deutsche Borreliose Gesellschaft) on the other hand [1, 2]. As a clinician that the author is, who witnesses the suffering of his patients on a daily basis, he appeals for the collaboration of all parties involved. He asks questions about the infection with borrelia, the possibility of a chronic Lyme disease, the existence of chronic infections generally in patients with weaker immune systems, the symptoms of an infection with borrelia (of different strains) and other tick-borne diseases, the sensitivity and specificity of the determination of antibodies in the everyday practice, the validity of EliSpot and the CD57 values for diagnosis, the treatment with antibiotics, the measures to control and support the patient's metabolism (adjuvants) before and during treatment, the phenomenon of erythema migrans, the biofilms that are complicating treatment and the other phenomenon of pleomorphic, slowly growing bacteria types (so called persister forms and L-forms) that may cause possible relapses . This contribution relies on a thorough study of literature. You will find a comprehensive list of references and links in the annex to prompt and inspire every reader interested in this topic. The author shares his thoughts and questions with the only purpose to stimulate factual discussions.
Key words: chronic Lyme disease, chronic borreliosis, late borreliosis, direct detection of borrelia, serology of borrelia, immunology, EliSpot, CD57 natural killer cells, borrelia, co-infections, erythema migrans, pleomorphic forms of bacteria, “persister” forms of borrelia, bacterial biofilms, antibiotic treatment, antibiotic companion diagnostics and antibiotic accompanying therapies.
Abstract / Summary ... 2
Index of tables ... 4
1. General information ... 5
2. Does chronic Lyme exist? ... 11
3. Symptoms of Lyme disease, a multi-systemic illness, and co-infectors ... 13
4. Direct Detection of Borrelia ... 17
5. Indirect Tests: The Detection of Antibodies ... 18
6. The EliSpot (Interferon-gamma-test) ... 21
7. CD57-NK-cells ... 22
8. Antibiotic Treatment ... 23
9. Accompanying diagnostics and supporting remedies during antibiotic ... treatments. 29
10. Erythema migrans (EM), Lymphocytoma & ACA, obligation to report ... 31
11. Biofilms, pleomorphic forms, persister forms ... 33
12. Discussion ... 34
Literature / Bibliography ... 38
Index of tables
Table 1: List of intracellular pathogens
Table 2: Sensitivity and specificity of serology
Table 3: Treatment strategies in Lyme borreliosis and co-infections
Table 4: Acting types of antibiotics
Table 5: Postulate of the comparative causes of infectious diseases
1. General information:
The following observations are meant to both inform and encourage reflection. These thoughts and questions have emerged from Internet research for literature, questions from patients in the everyday practice and numerous conversations. As it appears, there is a great demand for conversation and information not only among the patients, but also among the colleagues who are confronted with these questions by their patients.
We know that there is an interdependency that exists between pathogens (viruses, bacteria, fungi, protozoa, parasites, etc.) and their corresponding virulence factors (or pathogenicity factor), and the immune system of the affected person, i.e. the host. Here, it is decisive in every case to identify if the affected person is only infected or if he or she really manifests the infection, i.e. if he or she is developing symptoms and how serious the disease is.
– It is known that some types of bacteria and viruses have developed mechanisms to modulate, bypass and mislead the immune system, in order to persist in the organism [4, 5, 6, 7]. We know that for some infections, a later reactivation of the pathogens is possible (e.g. herpes-simplex-virus, HIV, varizella-zoster-virus, Epstein-Barr-virus, TBC, etc.). We also know that a possible latent, sub-clinical infection with borrelia and its reactivation is not strictly excluded anymore [8, 78].
– It is known that some types of bacteria are capable of creating, within their own bacterial ecosystem, pleomorphic forms [9, 10] and biofilms [11, 12, 79]. Bacterial and viral pathogens can modulate T-lymphocytes and induce insensitivity (anergy) [4, 5, 6, 7]. Some pathogens are able to change their antigens and receptors on the surface, to manipulate and are resistant to the complement system of the affected person, or even to ‘mislead’, for instance, the cellular immune system [13, 9]. Those mechanisms also exist undeniably with borrelia bacteria [10, 102, 103, 135].
– First question: Therefore, is it not possible that some auto-immune diseases [15, 80, 81], allergies  or endocrine diseases (malfunctions) can emerge? Normally, immune processes are triggered, pushed and maintained. Then, they must somehow be reduced and stopped. What if the permanent activation and/or disorder of the immune system are also caused by persistent bacterial pathogens or special forms of them?
– It is also known that some pathogens can act upon the cerebral metabolism in order to cause dysfunction, for instance, in the metabolism of neurotransmitters (serotonin, dopamine, and noradrenalin). What consequences would this have on the treatment of some cases of depression, psychosomatic, psychiatric, neoplastic and endocrinological illnesses, as for instance the thyroid [16, 17, 18, 32, 35, 41, 42, 82]?
– There are also many more factors that have a negative effect on our immune system, such as environmental influences [19, 20, 21, 82], specific food products, toxins, heavy metals , mould, softeners (in toys and furniture, etc.). In this context, it is worth reflecting on the origin of our food and the conditions under which they were produced and processed (e.g. antibiotics in animal breeding, etc.). What methods (e.g. the tanning of leather using chromium) or what dyeing processes are used in the manufacture of clothes? There are many more environmental-medical factors that cannot be discussed in depths in this paper.
– In cases of highly stressful situations and conflicts, it is known that our immune system may shut down (or weaken) due to a chronic overproduction of stress hormones such as adrenalin and cortisol. We can also include stress that the patient puts on himself, any disturbance in the biological rhythm (e.g. night shifts), noise and infrasound [12, 21].
– Does our food still contain all the necessary minerals and vitamins [22, 23]? With our busy lifestyle today who actually manages to feed themselves a balanced diet?
– As mentioned before, we are familiar with the fact that some borrelia strains have the skill to modify or are resistant against the complement system [13, 102, 103] of the affected person. The complement system is an important part of the innate immune system. This skill appears to be one important virulence factor of borrelia. A new study shows that this skill is distinctively strong depending on the borrelia strain . We can deduce the following ideas from this:
A person becomes ill if the particular borrelia strain is resistant against his/her complement system. If the borrelia strain is not resistant to the complement system, then the patient is only infected but not sick. In both cases, we will be able to prove the presence of antibodies in the blood if the test method is sensitive enough.
The debilitated immune system, which results from this resistance against the complement system, is only a partially and functional weakness. There are no test methods to the date which can measure this; however, it does exist [13,102,103,135].
The weakening of the immune system caused by borrelia is not as strong as for HIV, as borrelia only affects one special part of the immune system. In the case of HI-virus, it is a massive and general weakening of the immune system that we can better measure and prove.
– Why cannot humans develope persistent immunity against borrelia?
– There are also interactions between the pathogens and strains .
These points of discussion mentioned above are regularly documented in many scientific studies of certain fields such as psychology, psychiatry, environmental medicine, occupational medicine, immunology, infection medicine and nutritional science [24, 25]. However, the interdisciplinary consideration of all these factors and influences as a comprehensive observation is missing in our understanding of disease processes.
What effect do all these influences have on the immune system? And how do they affect the patient’s capability to fight infectious pathogens? The patient’s future depends on the balance between the genetic configuration, the environmental influences upon his immune system and the factor of pathogenicity [25, 26].
Could it not be possible that chronic infections, chronic multi-system and multi-organ illnesses have several (multifactorial) causes, which result in the infected person actually becoming ill from the infections?
Therefore, do we not need individual, multi-modal and holistic therapeutic approaches?
It is unfortunate that the possibility of a chronic (bacterial, viral, fungal, mould) infection is generally not taken into consideration when we are presented with chronic complaints with unclear genesis (or cause). This is why we have gathered a wider range of literature in the annex (see Bibliography: e.g. for Chlamydia: pp. 78-81; for Ehrlichia/Anaplasma: pp. 88-91; for Yersinia: pp. 96-98; for Mycoplasma: pp. 98-99, for Bartonella: pp. 100-109; for Babesia: pp. 109-113).
A few thoughts about the ticks as the vector for tick-borne diseases:
The proliferation of infected ticks depends on temporal, climatic and geographical variations. In this sense, the living environment of the various wild animals and (already modified) immigration routes of some birds have to be considered. All of these animals are hosts for borrelia (of different strains). In the spreading of infected ticks by birds, we may focus on birds living mainly in grass, hedges and undergrowth. There the birds get the ticks and then starts the transport and distribution [106-112]. It seems that also dogs are interesting and other animals. Furthermore, we wonder if due to climate changes, more ticks and pathogens may be able to survive in different (new) regions.
Furthermore, our travel behaviour in this new globalised world also favours the spreading of infected ticks (by means of transport, clothing, and luggage).
A further borrelia strain was recently discovered as a human-pathogenic, the borrelia miyamotoi. The current serological test procedures are not capable of detecting this strain. It is a matter of fact that this borrelia strain was transported and worldwide distributed by birds as well. Who knows how many more undiscovered human-pathogenic borrelia strains or other tick-borne pathogens there are ?
Since there is often a lack of vitamins, minerals and trace elements, a control test is useful - at least of fat-soluble vitamins (vitamins A, D, E and K) and B6 and B12.
The importance of vitamin D comes more and more to light [113, 114]. It is important not only for the bone metabolism but also for the immune system, nerve system, and maybe also as a protection against cancer and/or chronic infectious diseases [27, 61, 69].
It may be common knowledge that the intestine is a big immunological organ , but do we actually know everything about it ? The addition of probiotics to the S3-guidelines for treating irritable bowel syndrome (IBS) shows just how important this topic is.
We know today that some patients have problems with their metabolism of food (absorption, processing and detoxification) and of medicine (keyword: cytochrome P450-enzymes and cascades, etc.). There are also indications that infections can directly influence the cytochrome P450 system . There are also such differences in the metabolism between men and women, children and adults .
Can a chronically progressing, multi-causal, multi-system illness successfully be treated, in all cases, with a short mono-therapy?
In this work, we concentrate mainly on intracellular pathogens.
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Table 1: List of obligatory and facultative intracellular pathogens
Intracellular pathogens are lipophilic (fat-soluble) and generally acid-resistant; as are the pathogens for tuberculosis and leprosy. Such pathogens use many mechanisms to escape the host’s immune system and antibiotics. In addition, they are capable of changing their appearance according to the milieu. They are pleomorphic and, therefore, cannot be identified easily. The spiral shaped borrelia does not “fit” into an erythrocyte (red blood cell), but the so- called persister form “fits” perfectly into the body cells that appear much bigger in comparison. Persister forms seem to exist “in vivo” also for borrelia; otherwise there would not be research about new strategies to treat them .
Another practical note: doxycycline is often used to treat Lyme disease. Officially, 200mg/d are recommended. Others apply 400mg/d. It is indeed possible to measure the doxycycline levels in the patient’s blood after around 3 or 4 days of the treatment, in order to verify that the prescribed 200mg/d is sufficient to place the patient within the therapeutic range. There are indeed patients who do not reach the therapeutic range with 200mg/d. We can also find out whether the patient takes the antibiotic correctly. The minimal inhibitory concentration of a drug (MIC) is defined in the laboratory using original bacteria, the so-called planktonic form of bacteria. In the case of pleomorphic forms of bacteria and for bacterial biofilms, the therapeutic range lies 10 to 1000 times higher than the minimal inhibitory concentration (MIC) of the drug.
The aim of the treatment is to be permanently free of symptoms. This way, we can avoid long term incapacities, repeated visits to the doctor, and hospitalisations with intensive diagnostic and unsatisfactory therapies, which focus mainly on the symptoms or the patient’s inability to work. As long as there is no solution to the suffering, patients will understandably continue to search. This is disadvantageous even for economical reasons .
In order to discuss such topics, it is important to admit these ideas:
– Not only viruses can cause chronic and/or reactivated infections (e.g. Herpes- viruses), but also bacteria, parasites and fungus (mould).
– Even patients who are not affected by the HI-Virus can have a deficient or weaker function of the immune system.
– The various chronic ailments of a patient can be triggered by several causes and/or infections simultaneously.
2. Does chronic Lyme exist?
Chronic (or late) stage Lyme disease is not denied anymore [10, 29]. However, it is believed to be a rather rare condition, described by only 3-5%. The question is from what and how did this figure come to exist. The official number of new cases of infections with Lyme disease in Germany per year is 60.000-100.000. A Medical insurance company in Germany has found many more (Techniker Krankenkasse found 789.000 new infections per year with Lyme Disease in Germany). And the CDC reported now (in 2015) about 300.000 new cases per year in USA.
It is important to note that there is a difference between chronic Lyme disease and chronic neuroborreliosis.
The general chronic progressive form (or “late form”) of the Lyme disease refers to a multi-system infection that goes far beyond the nervous system. It also affects organs and organ systems [24, 120, 121].
Chronic neuroborreliosis, on the contrary, only refers to manifestations of an illness in the nervous system. All parts of the nervous system can be affected (in various degrees). To be more specific: the central nervous system, the cranial nerves, the peripheral and the autonomous nerve system can be affected. Patients may describe varying pain appearing in various locations and intensities throughout the nervous system.
Unfortunately, there is no objective measurement method to those ailments. We rely on the description of the external appearance, with the corresponding façade, to diagnose a neurological or psychological illness, anxiety disorder, rheumatism, etc. . A search for possible causes, such as chronically progressing infections, is usually not carried out.
The frequency of chronic borreliosis (late stage) and active co-infections is not known. The commonly encountered affirmation that a patient cannot be affected by chronic borreliosis because their cerebrospinal fluid test was inconclusive ("exclusion borreliosis"), is not in every case right.
The frequency of chronic neuroborreliosis is also not known. The figures could change if we would test more frequently for it, and if the testing method for antibodies were more comprehensive, more specific and more eventually more sensitive. Maybe it is true that not all pathogenic borrelia strains and all causes for co-infections are known and detectable .
We can find up to 700 peer- reviewed studies that acknowledge the existence of a chronic borreliosis and co-infections. The collection of studies you can find on www.ilads.org. Are these studies de facto all wrong? Above all, if some of these studies were carried out by independent authors (neither IDSA nor ILADS members) and even by IDSA members?
We believe that every human being should be worth at least that much that possible illnesses due to infectious pathogens are considered, even Lyme-borreliosis and all other already known co-infections.
3. Symptoms of Lyme disease, a multi-systemic illness, and co-infectors
The following recommendations are meant for patients whose symptoms could not be entirely explained by previous diagnostics. A predetermined and thorough differential diagnostic is essential. It is not always or only Lyme-Disease. It could be also another infection as you can see in the bibliography. A patient can also have two or more infections at the same time. Also other infections and other tick-borne diseases can cause complaints [30, 31, 126] (see also Bibliography, pp. 78-133). We should consider and verify this. It may appear in living beings much more often than what we think.
We recommend considering a possible chronic infection in the case of an unclear diagnosis and/or if the symptoms are the following:
– Unexplained physical exhaustion (chronic fatigue syndrome – CFS, Encephalomyelitis) 
– Ambulant pain in muscles and articulations 
– Erratic pain 
– Neuropathic symptoms of undefined genesis - Unclear (poly-) neuropathies
– Atypical MS, MS-like symptoms 
– Fibromyalgia of undefined genesis 
– Some auto-immune diseases (e.g. seronegative rheumatoid arthritis) [15, 80, 81]
– Neurological and psychiatric issues of undefined genesis [16, 17, 41, 90]
– Chronic abdominal pain 
The question is: how or why are these symptoms or syndromes triggered ? We should not just name and treat these physical ailments, but try to find their origin in order to treat them adequately.
It is known that a chronic or late-stage disease can appear very differently from an acute stage, be it clinically or in laboratory values. The difficulty lies in the chronic or late-stage manifestations.
We also know that different borrelia strains (and also leptospira strains) can generate a variation of symptoms. Also we do know that a patient can be infected with several borrelia strains and other activated pathogens. A tick can carry and transmit more than one human-pathogenic borrelia strain simultaneously and other tick-borne diseases.
Infections with borrelia and co-infections (or accompanying infections) can manifest themselves as disease in the eyes , the nervous system [16, 17, 37, 77, 83], the gastrointestinal tract [36, 28], the urinary tract , the blood vessels , the heart , the connective tissue, the articulations, the lung and the liver-gall system .
- When confronted to migrating episodic joint pains, do we consider chlamydia trachomatis, chlamydia pneumonia , Yersinia , borrelia, mycoplasma spp.  etc. for a possible cause (see Bibiliography, pp. 78-98)?
- Could a seronegative rheumatoid arthritis (RA) or a seronegative borreliosis in fact be a chlamydosis, mycoplasmosis or a yersiniosis, or even a borreliosis with a yet undiscovered borrelia strain  (see General Information, page 5)? Unfortunately, chronic forms of infections are up to now often not accepted as a cause of illness.
- When treating chronic and/or relapsing rhinitis, sinusitis, bronchitis or dry cough during weeks, do we think - after the exclusion of a malignancy - of chlamydia pneumonia, mycoplasma pneumonia, legionella, aspergillus, etc.?
- Is there a chronic chlamydia-infection , especially in people that have a weakened immune system? And can it really be treated sufficiently with three days of Azithromycin?
- Is it true that it is only people with diabetes mellitus, alcohol-based illnesses, tumours, chemotherapies, organ transplants, genetic immune defects and the elderly that suffer from a weakened immune system and that all the others are considered immune-competent? Could it not be possible that someone with a weakened immune system due to chronic infections and the already mentioned environmental influences (see General Information) could fall ill and display symptoms?
- There are many diabetic patients. Do we think about the known immunodeficiency in these patients and the possibility that they can have a chronic infection if they show unclear symptoms or even poly-neuropathy?
- What is the current research situation regarding the origins of some auto- immune diseases, allergies and endocrine disorders? Do we consider in respect to those symptoms the possibility of infections (chlamydia, mycoplasma, ehrlichiosis, bartonellosis, rickettsiosis, neo-ehrlichia, etc.) [5, 51, 53, 75, 76, 83, 84, 89, 91, 92, 93. 94, 95, 96, 97]?
- When dealing with Chronic Fatigue Syndrome (CFS) , a chronic viral infection is discussed as a cause. Could it not be that chronic bacterial infections and/or also fungal and mould infections are added to it?
- Do we check for borrelia, chlamydia or other infectious pathogens in cases of transient- ischemic attack (TIA) or apoplectic strokes (especially in younger patients) since they can also cause vasculitis? 
- There exists a significant literature on neuroborreliosis, on the so-called co- infections and on bacterial causes in Encephalomyelitis disseminate (Multiple Sclerosis, MS)  and Alzheimer [41, 122, 123] (see Bibliography, pp.60-64).
- A recent case study in the USA has shown the deadly outcome of Lyme- Carditis in a 17-year-old boy. During the autopsy, the pathologists have found borrelia directly in the tissue of the heart muscle . This allowed a direct detection of the pathogen. How often does an autopsy take place under such suspicion and how many pathologists think about it? However, this is the only way to understand this disease better.
- Just a few words about fibromyalgia: Based on our researches there is no controversy about the fact that Borrelia can cause neuropathic complaints. There is a study which has shown that the pains in fibromyalgia are neuropathic pains (neuropathic nature) . Therefore, isn’t it possible that patients with fibromyalgia could suffer from a Borrelia infection?
Inspite of showing objective signs of inflammation such as swelling of the joints, erythemas, swellings, pain and sub-febrile temperatures, inflammation parameters such as blood sedimentation rate (ESR) and C-reactive protein (CRP) are usually unobstrusive in Lyme-borreliosis.
In the following chapters, we describe some existing detection methods for pathogens. However, all detection methods in regards to borreliosis and co-infections have their qualitative limitations, esp. all indirect methods. None of these methods are sufficiently convincing when used separately. In order to get a trustworthy diagnosis, several detections and indication methods must be used at the same time.
4. Direct Detection of Borrelia
Without a direct detection of pathogens all discussions about Lyme disease are made difficult. It often becomes a very emotional discussion. The chances to detect directly a pathogen are higher when taken from tissue (as it was shown with the examination of the heart muscle in the case of Lyme-Carditis) . Certainly, this is not possible in everyday diagnosis.
Available to us are some accredited borrelia direct detection tests through patented borrelia culture + PCR (polymerase chain reaction) and the borrelia Focus Floating Microscopy + immune-histo-chemistry .
Both procedures are laborious and costly. Also, according to our knowledge, they alone are not sufficiently reliable. The direct detection methods for borrelia are not considered to be routine procedures due to financial reasons.
Other tests, such as proteome-determination or the detection of electromagnetic signals (EM), are not always recognised and mainstream .
Unless there is a direct detection of pathogens, a scientific discussion about chronic Lyme-borreliosis will remain controversial and a bone of contention.
There are two areas which we turn to regarding indirect testing methods of a patient's immune system. On the one hand, there is the humoral immune system which produces antibodies - which we test in serum (e.g. ELISA, CLIA, Immunoblot). On the other hand, there is the cellular immune system, the cytology (e.g. EliSpot, CD57 natural killer cells, Th1/Th2 balance).
5. Indirect Tests: The Detection of Antibodies:
Determination of antibodies maps the humoral immunological response. In doing so, we can observe what actions the immune system is undertaking against the pathogen. This works only under the condition that the immune system has indeed reacted and that the test procedures are sensitive and specific enough to measure this immune response.
To evaluate the activity of the humoral immune system, we measure activity, quantity and type of antibodies (e.g. ELISA [44, 50], CLIA, Immunoblots [45, 50]).
It is important to mention the fundamental problem of the general accepted standardisation, i.e. the sensitivity of the testing methods. All laboratories testing blood or cerebrospinal fluid do not work with a standardised antibody system, but various and different test systems. As a consequence, the same blood and cerebrospinal fluid samples can result in different findings (negative, borderline or positive) when analysed in different laboratories. These deviations can be incredibly large in blood and also in CSF (see Bibliography, pp. 69-72 and page 121).
If inadequate antigens are used, then the present antibodies cannot be measured. In this case, the test is a false negative and the patient is erroneously diagnosed as “not infected”. Generally, the following should be asked: Do we look in the right “place” (sample) with the right “means” (antigens) for the right pathogens? In the case of borrelia miyamotoi, the determination of antibodies would, at present, be negative .
It is also crucial that only very sensitive test systems should be used. The question is: how good (or “bad”) is the sensitivity (or “cut-off-level”) of the used antibody test system? In other words, the sensitivity is not only conditioned by the manifestation and duration of the illness, but also by the sensitivity of the used antibody test system (see below), and evidently also on the experience of the laboratory. Dr. Brian Fallon (an independent author) has confirmed in 2015 that all these problems still persist in the serological diagnostic for Lyme disease [133, see Bibbliography, page 121].
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Table 2: Borrelia-Westernblot plus Borrelia-ELISA-tests: indications of sensitivity and specificity.
The laboratory result is often used as a general basis for a diagnosis. The diagnosis, clinically and practically, generally rests upon the laboratory results only. This means that if the result is negative (e.g. when using a not so reliable test system in the laboratory), then the infection is excluded. This consequence is not in every case right.
Only clinicians and practitioners, who are in close contact with the patient, can decide what place the laboratory result has in the search for a diagnosis. Laboratory results are only an aid when making a diagnosis.
The clinic, the symptomatic profile, the recognition of an ailment pattern and the development of the illness are fundamental parameters for a diagnosis. We too, give the assessment of the ailments a much higher value.
There are many other diseases that are diagnosed on symptoms alone. No laboratory tests are necessary (or available) for such illnesses; this is the case for “seronegative” rheumatoid arthritis (RA), multiple sclerosis (MS), schizophrenia, fibromyalgia, depression, M. Alzheimer or M. Parkinson. Why is it impossible to have a “seronegative” borreliosis? Why is a diagnosis, in these conditions and using only clinical observations (or symptoms) not sufficient in the case of chronic Lyme disease?
There is one more question to a highly discussed topic: could the IgM-persistence in chronically developing illnesses not be a sign of a constant reactivation of the infection, instead of just being the usual and known marker for an early stage of the illness?  (see Bibliography, page 72 ).
An additional question in this chapter: Could it be that there are also problems with the sensitivity and specificity of the antibody-test-systems for co-infectors (and not only for borrelia) and that these co-infections can’t be detected in all patients ?
At the present there is a consensus that the antibody tests and also the progress of antibody titer can’t deliver information about the activity of the infection.
Hereto celluar immunology tests could eventually help. Until now there is one standardised celluar testing system or technique available, this is the EliSpot technique respectively the Interferon-Gamma test.