WO2016163888A1

WO2016163888A1 - Method and means for increasing antibodies against borrelia burgdorferi and increasing antigen of borrelia burgdorferi.

Info

Publication number: WO2016163888A1
Application number: PCT/NL2016/050252
Authority: WO
Inventors: Dennis TIMMERMANS
Original assignee: Pld Biochemistry B.V.
Priority date: 2015-04-10
Filing date: 2016-04-11
Publication date: 2016-10-13
Also published as: NL2014614B1; NL2014614A

Abstract

The present invention is directed to a method for the detection of Borrelia burgdorferi. The invention also relates to the use of a composition in a method for the detection of Borrelia burgdorferi. The invention further relates to a composition which increases antibodies against Borrelia burgdorferi as well as increases the antigens of Borrelia burgdorferi. Alternatively, the invention relates to methods and means for counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

Description

Method and means for increasing antibodies against Borrelia burgdorferi and increasing antigen of Borrelia burgdorferi. FIELD OF THE INVENTION

The present invention relates to the field of diagnosing Lyme disease. More particular, the invention relates to an improved method for detecting Borrelia burgdorferi. The invention also relates to the use of a composition in such a method. The invention further relates to a

composition which increases the antibodies against Borrelia burgdorferi as well as increases the antigens of Borrelia burgdorferi. Alternatively, the invention relates to methods and means for counteracting, controlling or reducing the symptoms of Lyme's disease in a subject. BACKGROUND OF THE INVENTION

Borrelia burgdorferi is a tick -borne spirochete which has been identified as the causative agent of Lyme disease. Lyme disease (Lyme borreliosis) is the most common vector-borne infectious disease in Europe, the United States and Asia. The disease is caused by at least three species of bacteria belonging to the Borrelia genus (Radolf & Samuels, 2010). In most cases, antibiotics eliminate the infection and its symptoms, especially if the illness is treated early. Delayed or inadequate treatment can lead to more serious symptoms, which can be disabling and difficult to treat.

The enzyme-linked immunosorbent assay (ELISA) test for the detection of antibodies to Borrelia burgdorferi is the most frequently used diagnostic method for the diagnosis of Lyme disease. However, the low sensitivity of the test is a frequent problem (Luger, 1990). The test is prone to false negative results when the concentration of antibodies in the serum of a test subject is too low or when the antibodies do not react well with the commercially produced antigens. Moreover, the test is prone to false positive results if the test subject has antibodies for a similar antigen non-related to Lyme Disease. The Dutch National Institute of Public Health and

Environmental Hygiene (RIVM) and the VU University Medical Center Amsterdam (VUmc) conducted a research study in 2011 to estimate the sensitivity of the ELISA test. It was estimated that the sensitivity of the ELISA test, performed by seven different laboratories on patients that were highly suspected of being infected with Borrelia burgdorferi, was between 34% and 59%, whereby in 36% of the cases where a Borrelia burgdorferi infection was present a false-negative result was obtained, indicating that the diagnosis of infection was missed altogether. The current low sensitivity of the diagnostic tests for Lyme disease highlights the need for means and methods that either increase the concentration of antibodies against

Borrelia burgdorferi in a test subject or increase the quality of commercially produced antigens, so as to increase the overall sensitivity of the

immunoassays for detecting Lyme disease. The present invention is directed to fulfilling the need of increasing the concentration of antibodies against Borrelia burgdorferi. On the basis of the inventive concept established by the present inventor, both a diagnostic as well as a therapeutic method is now proposed. SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method for typing a sample for the presence or absence of antibodies against Borrelia

burgdorferi, comprising the steps of a) providing a sample of a subject infected, or suspected of infected, with Borrelia burgdorferi; b.) determining a ratio of manganese to iron in said sample; c) detecting antibodies against Borrelia burgdorferi using an assay for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme-Linked Immuno Sorbent Assay (ELISA) or Western blot, if said ratio is higher than 1; and d) typing said sample for the presence or absence of antibodies against Borrelia

burgdorferi. The step b) of determining a ratio of manganese to iron in said sample is preferably a step wherein the amount of iron and manganese is determined in a body fluid of a subject, preferably blood or serum.

In another aspect, the invention provides a method for typing a sample for the presence or absence of antibodies against Borrelia

burgdorferi, comprising the steps of a) providing a body sample of a subject that is infected, or that is suspected of being infected, with Borrelia burgdorferi; and determining the ratio of manganese to iron in said sample; b) providing the same or a different body sample from said subject and performing on said sample an assay for the detection of antibodies against Borrelia burgdorferi, preferably by an Enzyme-Linked Immuno Sorbent Assay (ELISA) or Western blot; and c) qualifying the outcome of the assay for the detection of antibodies in step b) as a reliable test result in case the ratio of manganese to iron as determined in step a) is higher than 1, or qualifying the outcome of the assay for the detection of antibodies in step b) as an unreliable test result in case the ratio of manganese to iron as determined in step a) is lower than 1, to thereby type said sample for the presence or absence of antibodies against Borrelia burgdorferi.

The step of determining a ratio of manganese to iron in a sample according to step a) is preferably a step wherein the amount of iron and manganese is determined in a sample of a body fluid of a subject, preferably a blood or serum sample. The amount of iron or manganese in serum or blood is a standard medical laboratory test that is well known to the skilled artisan in the field of clinical chemistry. Preferably in order to determine the iron to manganese ratio, the Serum manganese concentration and the serum iron concentration are measured.

In a preferred embodiment of a method for typing according to the invention, if said ratio in said sample is equal to or lower than 1, said method further comprises the steps of e) administering, preferably orally, manganese and/or an iron-sequestering compound, preferably acetyls alicy he acid, to said subject; and f), optionally, determining a ratio of manganese to iron in said sample; wherein steps e) and f) are performed after step b) and before step c).

The skilled person will understand that when the iron and manganese concentrations are measured in other body samples, their relative ratio may vary with that of the manganese to iron threshold value of 1.0, at which the testing for B. burgdorferi antibodies in the blood or serum, or other body fluid of the subject is best performed, since these antibodies are much more difficult to detect at manganese to iron ratios below 1.0.

Without wishing to be bound by any theory, it is believed that in a method of the invention the redox capability of Borrelia burgdorferi spirochetes is temporarily increased as a result of a relative increase in the level of manganese as compared to the level of iron. Through the increase in redox capability, Borrelia burgdorferi spirochetes may more effectively neutralize the free radical's attack from leukocytes. Through the increase in redox capability, Borrelia burgdorferi spirochetes may not only survive the free radicals' attack from leukocytes, but also multiply. Therefore the population of spirochetes within a subject is increased. As a result of the multiplication of the population of Borrelia burgdorferi spirochetes, antibodies against Borrelia burgdorferi are increased.

burgdorferi, comprising the steps of a) providing a sample, preferably a blood sample, of a subject infected, or suspected of being infected, with

Borrelia burgdorferi; wherein in said subject the manganese concentration has been increased through administering, preferably orally, manganese and/or wherein in said subject the iron concentration has been decreased through administering, preferably orally, an iron-sequestering compound, preferably acetyls alicy lie acid; b) detecting antibodies, against Borrelia burgdorferi using an assay for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme-Linked Immuno Sorbent Assay (ELISA) or Western blot; and c) typing said sample for the presence or absence of antibodies against Borrelia burgdorferi.

In a preferred embodiment of a method for typing as recited in the previous paragraph, in said subject the manganese concentration has been increased and/or in said subject the iron concentration has been decreased so as to cause in the blood of said subject a ratio of manganese to iron higher than 1.

In a preferred embodiment of a method for typing according to the invention, said manganese and/or iron-sequestering compound are in a composition.

In still another aspect, the invention provides a method for increasing the sensitivity of an immunoassay for detecting Borrelia burgdorferi in a subject, the method comprising a) providing a sample, preferably a blood sample, from a subject infected, or suspected of being infected, with Borrelia burgdorferi; wherein in said subject the manganese concentration has been increased through administering, preferably orally, manganese and/or wherein in said subject the iron concentration has been decreased through administering, preferably orally, an iron-sequestering compound, preferably acetyls alicy lie acid; b) detecting antibodies against Borrelia burgdorferi using an assay for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme-Linked Immuno Sorbent Assay (ELISA) or Western blot.

In a preferred embodiment of a method for increasing the sensitivity of an immunoassay according to the invention, in said subject the manganese concentration has been increased and/or wherein in said subject the iron concentration has been decreased so as to cause in the blood of said subject a ratio of manganese to iron higher than 1. In another aspect, the invention provides use of manganese and/or an iron-sequestering compound, preferably contained in a composition, in a method for typing or method for increasing the sensitivity of an

immunoassay according to the invention.

In still another aspect, the invention provides a composition comprising manganese and an iron-sequestering compound.

In a preferred embodiment of a combination according to the invention, the iron-sequestering compound is acetyls alicy lie acid.In another aspect, the invention provides the use of manganese and/or an iron- sequestering compound, as a supplement in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method of the invention.

In a further aspect, the invention relates to manganese and/or an iron-sequestering compound for use as a pharmaceutical compound in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method of the invention.

In another aspect, the invention provides a combination or supplement, preferably dietary or pharmaceutical, comprising manganese and an iron-sequestering compound, optionally further comprising one or more (pharmaceutically) acceptable excipients.

In a preferred embodiment of a combination or supplement according to the invention, the iron-sequestering compound is acetyls alicy he acid.

In another preferred embodiment of a combination or supplement according to the invention, the combination or supplement, when

administered to a subject, establishes in said subject, preferably in the blood of said subject, a ratio of manganese to iron higher than 1.

In an even further preferred embodiment of a combination or supplement according to the invention, the combination is for use in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method of the invention.

In another aspect, the invention provides a kit of parts comprising manganese and an iron-sequestering compound, said manganese and iron- sequestering compound optionally formulated with one or more

(pharmaceutically) acceptable excipients, for use in detecting Lyme's disease, preferably said detection of Lyme's disease involving performance of a method according to the invention.

In another aspect, the invention provides a combination or supplement, preferably dietary or pharmaceutical, comprising iron and optionally manganese, the combination optionally further comprising one or more (pharmaceutically) acceptable excipients.

In a preferred embodiment of a combination or supplement comprising iron, the combination or supplement comprises 50-30000 microgram of iron, preferably ferrous iron.

In a further preferred embodiment of a combination or supplement comprising iron, the combination or supplement further comprises 1-3000 microgram of manganese, wherein the weight ratio of iron to manganese, measured in the same unit of weight, is at least 3: 1.

In a further preferred embodiment of a combination or supplement comprising iron, the combination or supplement further comprises one or more compounds selected from the group formed by, or consisting of, ascorbic acid, methylsulfonylmethane (MSM), magnesium, folic acid, vitamin B12 (cobalamin) and/or cysteine.

In another aspect, the combination or supplement comprising iron as defined hereinbefore is for use as a medicament.

In another aspect, the combination or supplement comprising iron as defined hereinbefore is for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject. In a preferred embodiment of a combination or supplement comprising iron for use in counteracting symptoms, the combination or supplement establishes, when administered to said subject, in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1.

In another aspect, the invention relates to iron, preferably ferrous iron, for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

In another aspect, the invention provides a container for holding a combination or supplement comprising iron as defined hereinbefore, the combination or supplement in the container having an overall iron to manganese weight ratio of at least 3: 1.

In another aspect, the invention provides a kit of parts comprising at least two foodstuffs, preferably different foodstuffs, for use in

counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, wherein the foodstuffs comprised in the kit have an overall iron to manganese weight ratio of at least 3: 1.

In a preferred embodiment of a kit of parts for use in counteracting symptoms as defined hereinbefore, the at least two foodstuffs, when administered to said subject, establish in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1.

In a preferred embodiment of a kit of parts for use in counteracting symptoms as defined hereinbefore, the at least two foodstuffs are selected from the group formed by, or consisting of, anise, apples, apricots, asparagus, avocado, basil, bell pepper, black beans, black pepper, broccoli, brussels sprouts, capers, carrots, cashews, celery, chard, cherries, chili pepper, chive, coriander leaves (raw), coriander seeds, couscous, cumin, curry powder, dark chocolate (preferably at least 70% cocoa) , dill, eggs, figs (preferably dried), grapes, green beans, ketchup, kidney beans, leek, lentils (red or green), meat (all meats), mint (peppermint, herb), mustard (prepared), oatmeal, oyster mushrooms, oysters (mollusk), pasta (white), pear, peas, potatoes (nb no sweet potatoes), pumpkin (cooked), pumpkin seeds, radicchio, raisins, rocket, rosemary, scallions, onions, sesame seeds, shrimp, soy sauce, spinach, sugarsnaps, thyme dried), tomatoes, wheat flour (white), yellow corn cereal.

In another aspect, the invention provides a foodstuff for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, wherein said foodstuff, when administered to said subject, establishes in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1. In another aspect, the invention provides a method of controlling or modulating the immune response against Borrelia burgdorferi in a subject infected, or suspected of being infected, with Borrelia burgdorferi, comprising the step of a) administering a combination or supplement of the invention, preferably in a Borrelia burgdorferi antibody-titer increasing, or Borrelia burgdorferi antibody-titer reducing amount, depending on the application. A method of controlling or modulating the immune response against Borrelia burgdorferi may include any of the embodiments or aspects of the invention as defined herein, and is aimed to include both diagnostic application (by increasing antibody titers against Borrelia burgdorferi in the body of a subject), as well as therapeutic application (by decreasing antibody titers against Borrelia burgdorferi in the body of a subject, to thereby improve a patients feeling of wellbeing)

The Examples of the present invention show that within a sample, a manganese (measured in nmol/1) versus iron ratio (measured in mol/l), and expressed as absolute values, lower than one or higher than one is decisive for the decrease respectively increase in the titer of antibodies against Borrelia burgdorferi. Partipants 1, 2 and 3 in the Examples continuously worked towards (or maintained) a manganese versus iron ratio within their blood lower than one. Due to a continuous manganese versus iron ratio within their blood lower than one, the ELISA IgG test results testing for antibodies against Borrelia burgdorferi in the Examples decreased. This decrease of antibodies seems to suggest that at the end of the test of Example 1, Example 2 and Example 3, either the virulence of Borrelia burgdorferi spirochetes decreased or the population of Borrelia burgdorferi spirochetes decreased or both at the same time decreased. A striking result in Example happened: although the manganese

concentration in the blood sample of participant 3 increased, the ELISA IgG test results of participant 3 decreased. This is in line with the invention since the manganese versus iron ratio within the blood of participant 3 is lower than one at the beginning as well as at the end of the test (and therefore most likely also during the test).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "acetylsalicylic acid", as used herein, refers to the acetylated derivative of salicylic acid; also known as aspirin

(TheFreeDictionary.com (1), 2015). The structural formula of acetyls ahcy lie acid is:

The term "administering", as used herein, refers to any method which in sound medical practice gives a dosage of a composition used in this invention to a mammal, where the method is for example, but not limited to, oral, intramuscular or intravenous administration. The method of

administration can vary depending on various factors, such as the composition and severity of the disease. Oral administration of the composition, combination, supplement, kit or foodstuff is preferred.

The term "antibody" , as used herein, refers to a mammahan, preferably human, immunoglobulin protein or any fragment thereof. The term "antibody", as used herein, preferably refers to a level, titer or concentration of antibodies, preferably immunoglobuhn G (IgG) and/or immunoglobulin M (IgM), present in a sample, preferably blood sample, of a mammahan, preferably human, subject. Known antibodies against Borrelia burgdorferi are of but not limited to the class immunoglobulin G (IgG) and/or immunoglobulin M (IgM) and specifically and/or selectively bind to an antigen of Borrelia burgdorferi.

The term "antigen", as used herein, refers to a molecule capable of generating an immune response, preferably an antibody response, in a mammahan, preferably human subject. In the context of the present invention, the term "antigen" particularly refers to antigens derived from Borrelia burgdorferi.

The term "Borrelia burgdorferi" , as used herein, refers to a bacteria that is the causative agent of Lyme disease. The term includes reference to, but is not limited to subspecies such as Borrelia burgdorferi s.s., Borrelia afzehi, Borrelia garinii, Borrelia valaisiana, Borrelia

lustianiae, Borrelia bissettii, Borrelia miyamotoi and other sensu lato Borellia species.

The term "composition", as used herein, refers to that of which a thing is composed or formed. Preferably, the term "composition", relates to a composition comprising manganese and another agent such as

acetyls ahcy lie acid.

The term "cytosol", as used herein, refers to the intracellular fluid inside cells. In the context of the invention, the term "cytosol" refers to the intracellular fluid of leukocytes. The term "detecting" , as used herein, refers to both the qualitative and quantitative measurements of a target molecule in the broadest sense.

Preferably, the term "detecting", refers to detecting for the presence or absence oiBorrelia burgdorferi. Detecting oiBorrelia burgdorferi can occur via but is not limited to an ELISA test and Western blot test, and preferably occurs by measuring the concentration of antibodies against Borrelia burgdorferi by ELISA and/or measuring the presence, and preferably the amount, of antibodies via Western blot. The skilled person knows how to perform these immunoassay tests.

The term "ELISA", as used herein, refers to an enzyme-linked immunosorbent assay which can be performed to detect either the presence of antibodies or the presence of antigens in a sample. Preferably, the ELISA test testing for Lyme disease, detects the presence of IgM antibodies against Borrelia burgdorferi and/or the presence of IgG antibodies against Borrelia burgdorferi. The skilled person understand that any ELISA test for detecting antibodies against Borrelia burgdorferi is envisaged in a method for typing according to the invention.

The term "ferritin", as used herein, refers to a protein that sequesters free cytosolic iron, the main catalyst of oxygen radical formation (Oberle et. al., 1998). The skilled person understands that ferritin can be used to decrease iron levels in the blood of a subject as well as decrease iron levels in the cytosol of macrophages of a subject.

The term "free radicals", as used herein, refers to chemicals containing atoms with an unpaired electron in its outer orbit.

The term "immunoassay", as used herein, refers to a biochemical test that measures the presence or concentration of a macromolecule in a solution through the use of an immunoglobulin such as an antibody.

Immunoassays employ a variety of different labels to allow for detection of antibodies and antigens. Labels are typically chemically linked or

conjugated to the desired antibody or antigen. The skilled person understand that any immunoassay suitable for detecting antibodies against Borrelia burgdorferi is envisaged in a method for typing according to the invention. Preferred immunoassays are ELISA and Western blot.

The term "iron", as used herein, refers to a common metallic element essential for the synthesis of hemoglobin. Its atomic number is 26 and its atomic weight is 55.85 amu. The term "iron", as used herein is commonly referred to as Fe. The term preferably refers to ionic iron, such as Fe⁺, Fe²⁺ and/or Fe³⁺.

The term "iron-sequestering compound", as used herein, refers to any compound that is able to directly or indirectly sequester iron, i.e. to reduce the concentration of free iron in a subject. For instance, it is well known that acetylsalicylic acid increases ferritin levels, of which it is well established that it reduces free iron levels. The term thus encompasses compounds having a iron-binding capacity sufficient to reduce free iron levels in a sample.

The term "leukocyte", as used herein, refers to immune associated cells derived from hematopoietic stem cells such as lymphocytes,

macrophages, neutrophils, eosinophils and basophils.

The term "macrophage", as used herein, refers to a white blood cell that engulfs and digests cellular debris, foreign substances and microbes in a process call phagocytosis. The term "macrophages", as used herein, includes reference to macrophages, monocytes and dendritic cells.

The term "manganese", as used herein, refers to a common metallic element found in trace amounts in tissues of the body, where it aids in the functions of various enzymes. Its atomic number is 25; its atomic mass is 54.938 amu (TheFreeDictionary.com (2), 2015). The term "manganese", as used herein is commonly referred to as Mn. The term preferably refers to ionic Mn, such as Mn⁺ or Mn²⁺, preferably Mn²⁺.

The term "manganese versus iron ratio in a sample", as used herein, refers to the manganese concentration (measured in nmol/1) divided by the iron concentration (measured in mol/l) in a blood sample: The manganese concentration versus iron ratio in a blood sample can be calculated by first measuring the manganese concentration in a blood sample (measured in nmol/1) and the iron concentration in a blood sample (measured in mol/l). The skilled person is well aware of suitable methods for measuring iron levels and manganese levels within a blood sample from a subject. For example: if the manganese concentration in a sample is 12 nmol/1 and the iron concentration in a sample is 24 mol/l, the manganese concentration versus iron ratio in a blood sample in this example is 0,5 (12 nmol/1 divided by 24 mol/l). The skilled person is provided with guidance on how to determine such a ratio in the Examples, wherein it is stated how the concentrations of manganese and iron were measured.

It is noted herein that the weight ratio of manganese to iron, or the weight ratio of iron to manganese, in a composition, combination, supplement, kit or foodstuff according to the invention is calculated by dividing the amount of manganese by the amount of iron, or vice versa, respectively. This ratio is, contrary to the manganese versus iron ratio in blood, calculated by dividing manganese and iron in the same unit of weight, for example both manganese and iron in milligram or microgram. Preferably, the composition, combination, supplement, kit or foodstuff, for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject according to the invention, establishes, when

administered to said subject, in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1. It was found that by carefully selecting foodstuffs on the basis of their manganese to iron ratio, a ratio of manganese to iron in blood of lower than 1 could be established.

The skilled person is aware of methods for measuring manganese and iron concentrations in a subject. As a consequence, he or she can easily establish what amount of manganese and/or iron in a composition, combination, supplement, kit or foodstuff according to the invention is needed to establish a manganese to iron ratio in a sample, preferably a blood sample, that is higher or lower than 1, depending on the reason for administration. Preferably the composition, combination, supplement, kit or foodstuff of the invention has a ratio of iron to manganese of at least 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8:1 or 10: 1. From this it follows that manganese may be present in a composition, combination, supplement, kit or foodstuff of the invention for counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, if the amount of manganese satisfies such the weight ratio of iron to manganese. It is noted herein that women, especially women in a menstrual cycle, are prone to fluctuations in blood iron levels due to for instance menstrual bleeding. In the context of this invention, it can be beneficial for such women to administer a composition, combination, supplement, kit or foodstuff of the invention having for instance an iron to manganese ratio of at least 6: 1 or 8: 1 to counter the negative effects of iron loss through menstrual bleeding on the symptoms of Lyme's disease.

The term "foodstuff, as used herein, includes both solid and liquid edible materials. The term includes reference to drinks such as health drinks, vegetables, fruits, soups, cereals and the like.

The term "supplement", as used herein, includes a substance or composition that comprises iron and optionally manganese. Preferably, the term refers to a feed or dietary supplement, preferably in the form of a pill. A supplement of the invention can be in the form of a single composition wherein both manganese and iron are co-formulated. Alternatively, in the context of detecting Lyme's disease, the term "supplement" includes a substance or composition that comprises manganese and/or an iron- sequestering compound, preferably in the form of a pill.

The term "combination", as used herein, includes reference to a combination of a substance or composition that comprises iron and a substance or composition that comprises manganese and/or an iron sequestering compound. Such a combination may be in the form of two separate pills, preferably comprised in a container of the invention, or as a kit of parts.

The term "overall iron to manganese weight ratio", as used in the context of a container of the invention or a kit of parts of the invention, refers to the total iron to manganese weight ratio formed by all substances, compositions or foodstuffs, comprising iron and/or manganese, that are comprised in a container of the invention or in a kit of parts of the invention. As stated hereinbefore, such a ratio is calculated by dividing iron and manganese in the same unit of weight, for example both manganese and iron in milligrams or micrograms.

The term "manganese super oxide dismutase", as used herein, refers to a gene that is a member of the iron/manganese superoxide dismutase family. The term "manganese super oxide dismutase" is also known as MnSOD, SodA or Sod2. Manganese super oxide dismutase which catalyzes the dismutation of superoxide anion (O^2~) to hydrogen peroxide (H²0²) and O². The skilled person knows that Borrelia burgdorferi MnSOD and human MnSOD are different. Human manganese super oxide

dismutase encodes a mitochondrial protein that forms a tetramer which is a protein with four subunits and binds one manganese ion per subunit. The term "Borrelia burgdorferi MnSOD", as used herein, refers to a Borrelia burgdorferi superoxide dismutase containing manganese which is an enzymatic defense against reactive oxygen species synthesized by dedicated enzymes in phagocytic cells like neutrophils and macrophages" (Nichols et. al., 2000; Kimball, 2015). Borrelia burgdorferi MnSOD forms a dimer which is a protein with two subunits and binds one manganese ion per subunit.

The term "sample", as used herein, refers to any sample from a biological source, including but not limited to blood samples, spinal fluids, a section of tissue obtained for example by surgery, plasma and serum samples. The preferred embodiment of this aspect of the present invention may be a body sample, preferably a blood sample, of a subject that is infected, or that is suspected of being infected, with Borrelia burgdorferi . The skilled person is well aware of suitable methods to take a blood sample from a subject, for example via venipuncture.

The term "subject", as used herein, refers to a mammal, preferably a primate, more preferably a human. In the context of the invention, the term "subject" preferably relates to a mammal, preferably human, suffering or suspected to suffer from Lyme disease or a mammal, preferably human, suffering or suspected to suffer from an infection with Borrelia burgdorferi.

The term "sensitivity", as used herein, refers to the ability of a test, preferably an immunoassay as described herein, to identify a condition correctly. Sensitivity may be calculated by dividing the number of true positives in a population by (the number of true positives plus the number of false negatives).

The term "increasing", used in the context of a method of increasing the sensitivity of an immunoassay according to the invention, refers to an increase in the level or titer of antibodies against Borrelia burgdorferi, or antigens of Borrelia burgdorferi, of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90 or 100% as compared to the level or titer of antibodies against Borrelia burgdorferi, or antigens of Borrelia burgdorferi, in a sample of a subject infected with Borrelia burgdorferi, which subject has not received, or did not yet receive, manganese and/or an iron-sequestering compound such as acetyls alicy lie acid.

The phrase "reducing the symptoms of Lyme's disease", as used herein, refers to the administration of a composition, combination, supplement, kit or foodstuff according to the invention so as to alleviate the symptoms of Lyme's disease in a subject. Preferably, the reduction or alleviation of symptoms is a decrease in the level or titer of antibodies against Borrelia burgdorferi of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 100% as compared to the level or titer of antibodies against Borrelia burgdorferi, or antigens of Borrelia burgdorferi, in a control sample of a subject infected with Borrelia burgdorferi, which subject did not receive, or did not yet receive, a composition, combination, supplement, kit or foodstuff according to the invention. The control sample is preferably obtained from the subject that is receiving said composition, combination, supplement, kit or foodstuff according to the invention. Alternatively, or in addition, a reduction or alleviation in symptoms of Lyme's disease can be measured for one or more symptoms selected from the group of (i) symptoms associated with the head, face and neck, including headache, facial paralysis, stiff neck, heightened allergic sensitivities, jaw pain, sore throat, swollen glands, (ii) symptoms associated with eyes/vision, including double or blurry vision, conjunctivitis, oversensitivity to light, eye pain, swelling around eyes, floaters/spots in the line of sight or red eyes, (iii) symptoms associated with ears/hearing, including decreased hearing, ringing or buzzing in ears, sound sensitivity or ear pain, (iv) symptoms associated with the digestive/excretory system, including nausea, vomiting, irritable bladder, unexplained weight loss or gain, (v) symptoms associated with the respiratory or circulatory system, including breathing difficulty, heart palpitations, heart block, hearth murmur or chest pain, (vi) symptoms associated with the musculoskeletal system, including joint and/or muscle pain, cramps, loss of reflexes, loss of muscle tone, muscle weakness, (vii) psychiatric symptoms, including mood swings, depression, personality changes, aggressive behavior/impulsiveness, overemotional reactions, disturbances in sleep such as too much sleep, too little sleep, difficulty falling or staying asleep, paranoia, obsessive-compulsive behavior, bipolar disorder, or schizophreny, (viii) symptoms associated with the neurologic system, including numbness, tingling, burning/stabbing sensations, weakness or paralysis of limbs, tremors, seizures, stroke, motion sickness, lightheadedness, encephalopathy, encephalitis, meningitis or

encephalomyelitis or (ix) cognitive symptoms, including dementia, memory loss or attention deficit problem. All these symptoms are linked to Lyme's disease as is for instance submitted by the Lyme Research Alliance.

Clinicians are aware of suitable tests to test for the abovementioned symptoms. The terms "reducing", "controlling" and "counteracting", as used herein in the context of symptoms of Lyme's disease, are used

interchangeably and can be replaced by one another.

The term "typing", as used herein, refers to determining whether a subject is infected with Borrelia burgdorferi. Preferably, typing occurs by determining the presence or absence of antibodies against Borrelia burgdorferi in a subject. If said antibodies are present, said subject is typed as being infected with Borrelia burgdorferi. The term "Western blot", as used herein, refers to the Western blot test, also called immunoblot, which separates proteins to their particle weights by using denaturing gel electrophoresis and then transferring these proteins electrophoretically (blotting) to a solid support matrix, which is generally a nitrocellulose (NC), or polyvinylidene fluoride (PVDF) membrane. The transfer of the proteins is followed by immunological detection of these proteins. For Lyme disease, the Western blot preferably detects IgM antibodies against Borrelia burgdorferi as well as IgG antibodies against Borrelia burgdorferi. A skilled person knows that different laboratories can use different antigens for detecting antibodies against Borrelia burgdorferi. The skilled person also understand that any Western blot test for detecting antibodies against Borrelia burgdorferi is envisaged in a method for typing according to the invention.

The present invention also discloses a method for treating Lyme's disease. In aspects of this invention, a method for treating Lyme's disease includes reference to counteracting, controlling or reducing the symptoms of Lyme's disease. Also included in a method of treating Lyme's disease is a method of controlling or modulating the immune response against Borrelia burgdorferi in a subject infected, or suspected of being infected, with

Borrelia burgdorferi. In the context of this specification, the terms "treatment" and "treating" refer to any and all uses which remedy a condition or disease or symptoms thereof, prevent the establishment of a condition or disease or symptoms thereof, or otherwise prevent or hinder or reverse the progression of a condition or disease or other undesirable symptoms in any way whatsoever.

In the context of this specification, the term "therapeutically effective amount" includes within its meaning a non-toxic amount of each of the indicated compounds, alone or in combination, sufficient to provide the desired therapeutic effect. The exact amount will vary from subject to subject depending on the age of the subject, their general health, the severity of the disorder being treated and the mode of administration. It is therefore not possible to specify an exact "therapeutically effective amount", however one skilled in the art would be capable of determining a

"therapeutically effective amount" by routine trial and experimentation.

Compounds used in aspects of this invention are suitably provided in the form of a pharmaceutically acceptable salt. In the context of this specification, "pharmaceutically acceptable salts" include, but are not limited to, those formed from: acetic, ascorbic, aspartic, benzoic,

benzenesulfonic, citric, cinnamic, ethanesulfonic, fumaric, glutamic, glutaric, gluconic, hydrochloric, hydrobromic, lactic, maleic, malic, methanesulfonic, naphthoic, hydroxynaphthoic, naphthalenesulfonic, naphthalenedisulfonic, naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic, p-toluenesulfonic, tartaric, trifluoroacetic,

triphenylacetic, tricarb ally lie, salicylic, sulfuric, sufamic, sulfanilic and succinic acid.

Borrelia burgdorferi and MnSOD

Almost all pathogenic bacteria require iron for growth. When infected with a pathogenic bacteria, a line of defense of the human body is withholding the necessary iron to prevent the pathogenic bacteria from growing which is named nutritional immunity. The most important form of nutritional immunity is the sequestration of nutrient iron (Kehl Fie & Skaar, 2010). Amongst others, the human body increases ferritin,

lactoferrin, transferrin, hepcidin, albumin which are iron-binding proteins to sequest er cytosolic iron as to effectively starve pathogenic bacteria from obtaining iron. The body also produces hepcidin that prevents iron from being absorbed in the gut and prevent iron coming in bloodstream

(ScienceDaily, 2015).

For a successful infection of humans, pathogenic bacteria must find a way to overcome this iron limitation within the host. Borrelia burgdorferi has successfully overcome this iron hmitation by substituting manganese for iron in metalloenzymes and therefore eliminating the requirement for iron (Cassat & Skaar, 2011). According to Dr Zhiming: "Out of the thousands of bacteria known, the Lyme disease agent and only one or two other bacterial species do not require iron for growth" (Guttenberg Hospital, 2009). Since the Borrelia burgdorferi bacteria require manganese and not iron for growth, the human body cannot effectively starve Borrelia burgdorferi spirochetes by lowering iron levels within the body of a mammal.

Another line of defense of the human body are leukocytes, which are the cells of the immune system that are involved in protecting the body against these pathogenic bacteria. Leukocytes attack pathogenic bacteria with free radicals (O^2-). Free radicals are radicals with an unpaired electron, which makes it highly reactive. Superoxide dismutase (SOD) is an

antioxidant enzyme with an ability to disproportionate the free radical (O^2-) into either ordinary molecular oxygen (O²) or hydrogen peroxide (H²O²). SOD's can be categorized in three different groups: 1) MnSOD/FeSOD, which binds either iron or manganese. 2) CuSOD/ZnSOD, which binds either copper or zinc and 3) NiSOD which binds to nickel. MnSOD/FeSOD's are used by prokaryotes, protists, and in mitochondria of eukaryotes. To fight off the free radicals attack from leukocytes, pathogenic bacteria can use an enzymatic antioxidant defense in the form of FeSOD and MnSOD or a combination of both (Wikipedia, 2015). Borrelia

burgdorferi bacteria only uses MnSOD as an antioxidant defense (Dafhne Aguirre et. al. 2013).

Preferred embodiments

The invention provides a method for typing a sample for the presence or absence of antibodies against Borrelia burgdorferi as well as a method for typing a sample for the presence or absence of antigens of

Borrelia burgdorferi. The invention may be delivered via embodiments but not limited to such as a device of determined physical form, such as a tablet, patch, capsule, pill or troche. The route of administration of manganese and/or an iron-sequestering compound includes, but is not limited to, oral, rectal or parenteral administration. Oral administration offers advantages over other routes of administration.

In a preferred embodiment, pills represent one form of a

composition of this invention comprising the inventive combination of manganese and acetyls alicy lie acid.

The invention further provides a combination, supplement or composition comprising manganese and/or an iron-sequestering compound such as acetyls alicy lie acid, said combination, supplement or composition optionally further comprising one or more (pharmaceutically or dietary) acceptable excipients. Suitable pharmaceutical excipients are inter alia described in the Handbook of Pharmaceutical Excipients, 7 Updated Edition of June 2012, by Rowe et al. Methods and means for manufacturing pharmaceutical formulations are inter alia generally described in the

Handbook of Pharmaceutical Manufacturing Formulations, second edition of 2009, six -volume set, by Niazi. These publications are incorporated herein by reference. When in the form of a single dosage unit, such as a pill, the composition may comprise about 175 mg acetyls alicy lie acid in combination with about 35 mg manganese, for instance acetyls alicy lie acid in the range 1 mg, 5, 20, 30, 40, 50, 60, 70, 80 or 90 to 100, 200, 300, 350, 375, 400, 450, 500, 600, 700, 800, 900 or 1000 mg acetyls alicylic acid and for instance manganese, preferably chelated manganese, in the range 0.1 mg, 0.3, 0.5, 1, , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 to 200 mg manganese. The dosage amounts may be given for an administration period up to four weeks.

Preferably, the single dosage unit is taken at least once, twice, trice or four times a day for a preferred fourteen days before undergoing an

immunoassay test. More preferably the single dosage unit is taken from about 45 to about 15 minutes before each meal, particularly preferable about 30 minutes before each meal. In addition to each meal, the single dosage unit can also be taken before going to sleep. Alternatively, or in addition, the dosage of manganese and/or an iron-sequestering compound is in a biologically effective amount, i.e. a Borrelia burgdorferi antibody-titer increasing amount. In other words, the dosage of manganese and/or an iron- sequestering compound is preferably in such an amount that the immune system of the subject is activated, resulting in an increase in antibody-titer against Borrelia burgdorferi. This latter effect may be designated as a diagnostically effective amount of the manganese and/or an iron- sequestering compound.

Preferably, in a combination or supplement of the invention, manganese is bound to a negative counter-ion to maintain electric

neutrality, so as to provide for a salt. A preferred negative counter-ion is sulphate. Alternatively, manganese is chelated, preferably amino-acid chelated. Preferably, the combination or supplement comprises manganese in the range of 0.1 mg, 0.3 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg to 200 mg manganese, or any range based on the values mentioned. Preferably, the combination or supplement comprises an iron-sequestering compound, preferably

acetyls alicy lie acid, in the range of 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg or 90 mg to 100 mg, 200 mg, 300 mg, 350 mg, 375 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1000 mg acetyls alicy lie acid, or any range based on the values mentioned.

In one preferred embodiment, the method for detecting antibodies against Borrelia burgdorferi is but not limited to an immunoassay. The immunoassay may comprise but is not limited to an ELISA, Western blot, direct immunofluorescence assay, indirect immunofluorescence assay or agglutination test. For use in an ELISA, the sample is preferably plasma or serum, although another biological fluid may be used.

In another preferred embodiment of the invention, antibodies against Borrelia burgdorferi are but not limited to immunoglobulin G and/or immunoglobulin M. The method of the present invention provides

unobtrusive and inexpensive means for enhancing antibodies against

Borrelia burgdorferi in a sample as well as enhancing antigens of Borrelia burgdorferi in a sample.

In yet another preferred embodiment of the invention, the method for typing relates to an ELISA test that is based on, but not limited to, a (Ce) synthetic peptide that reproduces the sequence of a 26-mer invariable region of the surface antigen VlsE of Borrelia burgdorferi, also referred to as C6 Lyme ELISA (C6 (B. burgdorferi) Lyme ELISA, Immunetics, MA, US, Catalog # DK-E352-096). An immunoblot has demonstrated that this third generation ELISA, which includes a mixture of recombinant antigens of Borrelia burgdorferi decreases the chance of false positive results

(Marangoni, 2005). In a preferred embodiment of the invention, the immunoblot utilizes the antigens which are advised by the medical guidelines of that particular continent or country. Since multiple Borrelia burgdorferi subspecies exist in Europe, a preferred embodiment of the present invention is an immunoblot test which utilizes recombinant antigens as for the detecting of antibodies against Borrelia burgdorferi. Preferably the following antibodies against Borrelia burgdorferi are detected in Europe:

· IgM antibodies against Borrelia burgdorferi: p l8, p22 OspC (which is the same as p24 OspC), p31 (OspA), p39 (BmpA), p58, p66 and p 100.

^• IgG antibodies against Borrelia burgdorferi: p l8, p22 OspC (which is the same as p24 OspC), p31 (OspA), p39 (BmpA), p58, p66 and p 100.

Since in the United States mainly Borrelia burgdorferi s.s. exist, a preferred embodiment of the present invention may be but is not limited to an immunoblot test comprising native antigens of Borrelia burgdorferi s.s. for the detection of the following antibodies against Borrelia burgdorferi s.s..

Preferably the following antibodies against Borrelia burgdorferi s.s. are detected in the United States:

· IgM antibodies against Borrelia burgdorferi s.s.: p24 (OspC), p39 (BmpA), p41 (Flaggelin)

^• IgG antibodies against Borrelia burgdorferi s.s.: p l8, p21 OspC (which is the same as p22 OspC and p24 OspC), p28, p30, (BmpA), p41 (Flaggelin), p45, p58 (not GroEL), p66, p93.

In a preferred method of typing of the invention, antibodies against Borrelia burgdorferi, or antigens of Borrelia burgdorferi, are detected.

Correspondingly, in such a method of the invention, a sample of a subject is typed for the presence or absence of antibodies against Borrelia burgdorferi or a sample of a subject is typed for the presence or absence of antigens of Borrelia burgdorferi. The preferred method for detecting antibodies against Borrelia burgdorferi and/or antigens of Borrelia burgdorferi is by using an immunoassay. The skilled person is aware of the fact that immunoassays can use an antigen, preferably immobilized on the immunoassay, said antigen serving as bait for an antibody against Borrelia burgdorferi, to identify said antibody against Borrelia burgdorferi or use an antibody, preferably immobilized on the immunoassay, said antibody serving as bait for an antigen of Borrelia burgdorferi, to identify said antigen of Borrelia burgdorferi.

In a preferred embodiment of the invention, manganese and iron concentrations are measured in a non-invasive method by using a blood sample. The iron levels within a blood sample are preferably measured in mol/l and the manganese levels are preferably measured in nmol/1. The manganese concentration in the blood sample expressed in nmol/1 is then divided by the iron levels in the blood sample expressed in mol/l to obtain the manganese versus iron ratio. The skilled person is well aware of suitable methods for measuring iron levels and manganese levels within a blood sample from a subject.

Alternatively, in another preferred embodiment of the invention, the manganese (measured in nmol/1) versus iron ratio (measured in mol/l) is calculated by dividing the manganese levels measured within the macrophage by the iron levels measured within the macrophage. The skilled person is well aware of suitable methods for measuring iron levels and manganese levels within macrophages.

In another preferred embodiment of the invention, an iron- sequestering compound as acetyls alicy e acid is used. The skilled person will understand that acetyls alicy lie acid is an anti -inflammatory agent against almost all pathogenic bacteria but not against Borrelia burgdorferi. The skilled person understands that acetyls alicy lie acid will elevate ferritin levels within the body which in turn will decrease free iron levels within the blood as well as decrease cytosolic iron within macrophages. Bacteria that use iron for growth and/or use FeSOD as an anti-oxidant defense against free radical's attack from leukocytes will have trouble finding their much needed iron. A bacteria which uses FeSOD as an anti-oxidant defense against free radical's attack from leukocytes needs iron for increasing their redox capability against free radical's attack from leukocytes through the formation of the active anti-oxidant enzyme Fe²⁺-FeSOD. The skilled person is aware that out of the thousands of bacteria known, Borrelia burgdorferi spirochetes do not require iron for growth and do not utilize FeSOD as an anti-oxidant defense against free radical's attack from leukocytes. Therefore the skilled person understands that acetyls alicy lie acid is not an antiinflammatory agent in the context of a Borrelia burgdorferi infection.

The invention further provides a method for increasing the ratio of manganese to iron in blood of a subject infected or suspected to be infected with Borrelia burgdorferi, the method comprising the step: a) administering manganese and/or administering an iron sequestering compound, preferably acetyls alicy lie acid, to said subject.

The invention further provides a method for increasing the concentration of antibodies against Borrelia burgdorferi in blood of a subject infected or suspected of being infected with Borrelia burgdorferi, the method comprising the step: a) administering manganese and/or administering an iron sequestering compound, preferably acetyls alicy he acid, to said subject.

Correspondingly, in such a method of the invention, a sample of a subject is typed for the presence or absence of antibodies against Borrelia burgdorferi or a sample of a subject is typed for the presence or absence of antigens of Borrelia burgdorferi. The preferred method for detecting antibodies against Borrelia burgdorferi and/or antigens of Borrelia burgdorferi is by using an immunoassay. The skilled person is aware of the fact that immunoassays can be based on an antigen of Borrelia burgdorferi or antibody against Borrelia burgdorferi immobilized on an immunoassay, serving as bait, capable of binding to an antibody against Borrelia burgdorferi or to an antigen of Borrelia burgdorferi, respectively. It is understood that, the sample on which the ratio of manganese to iron is determined, can also be used for performing an assay for the detection of antibodies against Borrelia burgdorferi. It is, however, clear to the skilled person that such an assay can also be performed on a different sample of the same subject, provided that such a second sample is obtained from the subject after it has received a Borrelia burgdorferi antibody-titer increasing amount of a composition, combination or supplement as indicated herein for the purpose of typing a sample.

In one embodiment, the outcome of the assay for the detection of antibodies in step b) is qualified as a reliable test result in case the ratio of manganese to iron as determined in step a) is higher than 1, or qualifying the outcome of the assay for the detection of antibodies in step b) as an unreliable test result in case the ratio of manganese to iron as determined in step a) is lower than 1, to thereby type said sample for the presence or absence of antibodies against Borrelia burgdorferi.

Treatment: Counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

In the context of counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, the invention provides a

combination, composition or supplement, preferably dietary or

pharmaceutical, and preferably in a therapeutically effective amount, comprising iron and optionally comprising manganese, preferably the latter in trace amounts, the combination or supplement optionally further comprising one or more (pharmaceutically) acceptable excipients. Such a combination, composition or supplement is preferably suitable for oral administration and may take the form of a pill.

Preferably, a combination, composition or supplement of the invention comprises 50-30000 microgram of iron. More preferably, a combination, composition or supplement of the invention comprises 500- 20000 microgram of iron. Most preferably, a combination, composition or supplement of the invention comprises 1000- 10000 or 1000-5000 microgram of iron. Alternatively, and preferably, a combination, composition or supplement of the invention comprises 2.000-3000 or about 2.500 microgram of iron. Preferably, in a combination, composition or supplement of the invention, the iron is ferrous iron (Fe²⁺). The skilled person appreciates that, in general in solid formulations, ferrous iron is bound to a negative counter- ion to maintain electric neutrality. A preferred negative counter-ion is sulphate.

A combination, composition or supplement of the invention preferably further comprises 1-3000 microgram, preferably 10-2000 microgram, more preferably 50-1000 microgram and most preferably 100 to 500 microgram or about 250 microgram, of manganese, preferably Mn²⁺. A preferred negative counter-ion of the manganese ion is sulphate. If manganese is present in a combination, composition or supplement of the invention, the weight ratio of iron to manganese (measured in the same unit of weight) is at least 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1. Preferably, the weight ratio of iron to manganese is at least 10: 1. Manganese is preferably co-formulated with iron in a combination, composition or supplement of the invention so as to provide the subject with an influx of manganese which is used as a co-factor in the defense against reactive oxygen and nitrogen species. For reasons specified herein, the amount of manganese formulated in a combination, composition or supplement of the invention should be lower than the amount of iron.

Further ingredients of a combination, composition or supplement of the invention, which were found to have a positive effect on parameters of Lyme's disease, are vitamin C (ascorbic acid); methylsulfonylmethane (MSM); magnesium, preferably in the form of magnesiumcitrate; vitamin B9 (folic acid); vitamin B 12 (cobalamin); and/or cysteine, preferably in the form of N-acetyl-cysteine (NAC). A most preferred combination, composition or supplement of the invention comprises (i) about 2500 microgram of ferrous iron in the form of ferrous sulphate, (ii) about 250 microgram of manganese in the form of manganese sulphate, (iii) about 250 milligram of ascorbic acid, (iv) about 750 milligram of methylsulfonylmethane, (v) about 200 mg of magnesium citrate, (vi) about 500 microgram of folic acid, (vii) about 500 microgram of cobalamin, and (viii) 250 milligram of N-acetyl-cysteine (NAC), optionally further comprising a pharmaceutically or dietary acceptable excipient.

Suitable pharmaceutical excipients are described in the Handbook of Pharmaceutical Excipients, 7 Updated Edition of June 2012, by Rowe et al. Methods and means for manufacturing pharmaceutical formulations are generally described in the Handbook of Pharmaceutical Manufacturing Formulations, second edition of 2009, six -volume set, by Niazi. These publications are incorporated herein by reference.

The skilled person appreciates that the further ingredients referenced herein may be processed in the body into a biologically active form, such as is the case for folic acid, the latter being processed into the biologically active compound folate. The further ingredients, as defined herein, also cover the biologically active or activated forms of these ingredients.

A combination, composition or supplement of the invention is preferably administered orally at least once a day. Preferably, a

combination, composition or supplement of the invention is administered three times a day, for at least 1, 2, 3 or 4 weeks. The combination, composition or supplement may also be provided by other means of administration, including by parenteral or rectal administration.

Pharmaceutical compositions include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), inhalation (including use of metered dose pressurised aerosols, nebulisers or insufQators), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.

Generally, an effective dosage of a combination, composition or supplement present in pharmaceutical and other compositions of the present invention is expected to be in the range of about 0.000 lmg to about lOOOmg per kg body weight per 24 hours; about 0.00 lmg to about 750mg per kg body weight per 24 hours; about 0.0 lmg to about 500mg per kg body weight per 24 hours; about O. lmg to about 500mg per kg body weight per 24 hours; about O. lmg to about 250mg per kg body weight per 24 hours, or about l.Omg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range of about l.Omg to about 200mg per kg body weight per 24 hours; about l.Omg to about lOOmg per kg body weight per 24 hours; about l.Omg to about 50mg per kg body weight per 24 hours; about l.Omg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about 20mg per kg body weight per 24 hours, or about 5.0mg to about 15mg per kg body weight per 24 hours.

Alternatively, an effective dosage of a combination, composition or supplement may be up to about 500mg/m². Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m², about 25 to about 350mg/m², about 25 to about 300mg/m², about 25 to about 250mg/m², about 50 to about 250mg/m², or about 75 to about 150mg/m².

The invention further provides a combination, composition or supplement of the invention for use as a medicament, preferably for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

Alternatively, the invention provides a combination, composition or supplement comprising iron, preferably ferrous iron, and preferably manganese, for use as a medicament, preferably for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject. The invention also relates to iron, preferably ferrous iron, for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

The invention also relates to a use of iron, preferably ferrous iron, in the manufacture of a medicament for counteracting, controlhng or reducing the symptoms of Lyme's disease in a subject.

The invention also relates to a method for counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, comprising the step of a) administering to a subject suffering from Lyme's disease, or suspected to suffer therefrom, a combination, composition or supplement comprising iron, preferably ferrous iron.

The invention further relates to a kit of parts for use in

counteracting, controlling or reducing the symptoms of Lyme's disease in a subject. Preferably, in a kit of parts for use in counteracting, controlhng or reducing the symptoms of Lyme's disease according to the invention, the at least two foodstuffs are selected from Table 1 and/or from the group formed by anise, apples, apricots, asparagus, avocado, basil, bell pepper, black beans, black pepper, broccoli, brussels sprouts, capers, carrots, cashews, celery, chard, cherries, chili pepper, chive, coriander leaves (raw), coriander seeds, couscous, cumin, curry powder, dark chocolate (preferably at least 70% cocoa) , dill, eggs, figs (preferably dried), grapes, green beans, ketchup, kidney beans, leek, lentils (red or green), meat (all meats), mint

(peppermint, herb), mustard (prepared), oatmeal, oyster mushrooms, oysters (mollusk), pasta (white), pear, peas, potatoes (nb no sweet potatoes), pumpkin (cooked), pumpkin seeds, radicchio, raisins, rocket, rosemary, scallions, onions, sesame seeds, shrimp, soy sauce, spinach, sugarsnaps, thyme dried), tomatoes, wheat flour (white), yellow corn cereal. The aforementioned foodstuffs have an iron to manganese weight ratio of at least 2:1, preferably at least 3: 1. The skilled person understands that a kit of parts for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject may also comprise some foodstuffs that have an iron to manganese weight ratio lower than 2: 1, as long as the overall manganese to iron ratio in the kit is at least 2: 1, preferably at least

3: 1. Compositions suitable for buccal (sublingual) administration include lozenges comprising compounds of the invention for use in any aspect as described in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising compounds of the invention for use in any aspect as described in an inert base such as gelatine and glycerin or sucrose and acacia.

Compositions comprising compounds of the invention for use in any aspect as described suitable for oral administration may be presented as discrete units such as gelatine or HPMC capsules, cachets or tablets, each containing a predetermined amount of compounds of the invention for use in any aspect as described, as a powder, granules, as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Compounds of the invention for use in any aspect as described may also be present in a paste.

When the compositions comprising compounds of the invention for use in any aspect as described are formulated as capsules, the compounds of the invention for use in any aspect as described may be formulated with one or more pharmaceutically acceptable carriers such as starch, lactose, microcrystalline cellulose, silicon dioxide and/or a cyclic oligosaccaride such as cyclodextrin. Additional ingredients may include lubricants such as magnesium stearate and/or calcium stearate. Suitable cyclodextrins include oc-cyclo dextrin, -cyclodextrin,Y-cyclodextrin, 2-hydroxyethyl- -cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3-hydroxypropyl- -cyclodextrin and tri- methyl- -cyclodextrin. The cyclodextrin may be hydroxypropyl-β- cyclodextrin. Suitable derivatives of cyclodextrins include Captisol® a sulfobutyl ether derivative of cyclodextrin and analogues thereof as described in US patent No. 5, 134, 127. Tablets may be prepared by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine compounds of the invention for use in any aspect as described in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant (for example magnesium stearate or calcium stearate), inert diluent or a surface active/dispersing agent. Moulded tablets may be made by moulding a mixture of the powdered compounds of the invention for use in any aspect as described moistened with an inert liquid diluent, in a suitable machine. The tablets may optionally be coated, for example, with an enteric coating and may be formulated so as to provide slow or controlled release of compounds of the invention for use in any aspect as described therein.

Compositions for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and which may include suspending agents and thickening agents. A parenteral composition may comprise a cyclic oligosaccaride such as hydroxypropyl- -cyclodextrin. The

compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use.

Compositions suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Such patches suitably comprise compounds of the invention for use in any aspect as described as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the compound.

Compositions suitable for transdermal administration may also be delivered by iontophoresis, and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable compositions may comprise citrate or Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M of compounds of the invention for use in any aspect as described.

Spray compositions for topical delivery to the lung by inhalation may, for example be formulated as aqueous solutions or suspensions or as aerosols, suspensions or solutions delivered from pressurised packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant.

Suitable propellants include a fluorocarbon or a hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, e.g. dichlorochfluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1, 1, 1,2-tetrafluoroethane, 1, 1,2,2,3,3,3-heptafluoro-n-propane or a mixture thereof. Carbon dioxide or other suitable gas may also be used as propellant. The aerosol composition may be excipient free or may optionally contain additional composition excipients well known in the art, such as surfactants e.g. oleic acid or lecithin and cosolvents e.g. ethanol.

Pressurised compositions will generally be retained in a canister (e.g. an aluminium canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.

Medicaments for administration by inhalation desirably have a controlled particle size. The optimum particle size for inhalation into the bronchial system is usually 1- 10 μιη, preferably 2-5 μιη. Particles having a size above 20 μιη are generally too large when inhaled to reach the small airways. When the excipient is lactose it will typically be present as milled lactose, wherein not more than 85% of lactose particles will have a MMD of 60-90 μιη and not less than 15% will have a MMD of less than 15 μιη.

Compositions for rectal administration may be presented as a suppository with carriers such as cocoa butter or polyethylene glycol, or as an enema wherein the carrier is an isotonic liquid such as saline.

Additional components of the compositions may include a cyclic oligosaccaride, for example, a cyclodextrin, as described above, such as hydroxypropyl- -cyclodextrin, one or more surfactants, buffer salts or acid or alkali to adjust the pH, isotonicity adjusting agents and/or anti-oxidants.

Compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vasoline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof.

Compounds of the invention for use in any aspect as described is generally present at a concentration of from 0.1% to 20% w/w, or from 0.5% to 5% w/w. Examples of such compositions include cosmetic skin creams.

The composition may also be administered or delivered to target cells in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Specific examples of liposomes that may be used to administer or deliver a compound formula (I) include synthetic cholesterol, 1,2-distearoyl-sn- glycero-3-phosphocholine, 3-N-[(-methoxy poly(ethylene

glycol)2000)carbamoyl]-l,2-dimyrestyloxy -propylamine (PEG-cDMA) and l,2-di-o-octadecenyl-3-(N,N-dimethyl)aminopropane (DODMA).

The compositions may also be administered in the form of microp articles. Biodegradable microp articles formed from polylactide (PLA), polylactide-co-glycolide (PLGA), and έ-caprolactone have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J Pharm Pharmaceut Sci. 3(2) 234- 258).

The compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and organic solvent or organic solvent mixtures. Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate. Compounds of the invention for use in any aspect as described may be added to the SAIB delivery vehicle to form SAIB solution or suspension compositions. When the formulation is injected subcutaneously, the solvent diffuses from the matrix allowing the SAIB-drug or SAIB-drug- polymer mixtures to set up as an in situ forming depot.

The co-administration of compounds of the invention for use in any aspect as described may be simultaneous or sequential. Simultaneous administration may be effected by compounds of the invention for use in any aspect as described being in the same unit dose as a therapeutic or other agent, or compounds of the invention for use in any aspect as described and the therapeutic or other agents may be present in individual and discrete unit doses administered at the same, or at a similar time. Sequential administration may be in any order as required.

All publications mentioned in this specification are herein incorporated by reference. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include

embodiments having combinations of all or some of the features described.

It should be understood that this invention is not limited to the particular methodology, protocols, etc., described herein and, as such, may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. EXAMPLES The following Examples explain the invention in detail.

In the Examples the mechanism of the invention is investigated. Whereas the composition of the invention increases the manganese versus iron ratio within a blood sample of a subject which increases the ELISA IgG test results, the Examples prove the opposite as well: if the manganese versus iron ratio within a blood sample is continuous decreases (or maintained) below one, the ELISA test results decrease accordingly.

To qualify for participation in the test from the Examples, a patient had to be infected with Borrelia burgdorferi for at least six months or more. Since participants suffered from a Borrelia burgdorferi infection for more than six months, the Borrelia burgdorferi ELISA test results from the Examples only focused on immunoglobulin G (IgG).

The second qualification criteria for participation in the test, was that a patient's latest ELISA test had to have at least a positive Borrelia burgdorferi ELISA IgG test result of 25 kU/1 in the year 2014 and/or 2015 (this ELISA IgG test is considered positive in case the test result is higher than 4 kU/1). Since different laboratories can give different results, all ELISA tests had to come from same laboratory. In this case only ELISA IgG test results from the laboratory "Medische Laboratoria Dr. Stein &

Collegae" qualified.

The third and last qualification criteria to be included in the test from the Examples, was that at the starting point of the test, the Borrelia burgdorferi ELISA IgG test results also had to have at least a positive test result of 25 kU/1. This ELISA test also was taken solely at the laboratory: "Medische Laboratoria Dr. Stein & Collegae". One would be participant received a negative test result at the starting point from the test and therefore was excluded from being in the Examples.

At the starting point of the test, blood samples were taken from the participants. A Borrelia burgdorferi ELISA IgG test was performed on the blood samples of the participants at the laboratory "Medische Laboratoria Dr. Stein & Collegae". The manganese and iron concentration of the participants was measured in the blood samples at the laboratory "Reinier Medisch Diagnostisch Centrum".

Also at the starting point of the test, participant 1, participant 2 and participant 3 received the dietary recommendations represented by the food lists of Tables 1-3, from which they were only allowed to eat from the recommended list and neutral list. What the participants did not know was that each food item on the recommended food list contained a manganese versus iron ratio of less than 0,33. The neutral food list did not contain any manganese and therefore the calculation of the manganese versus iron ratio was unnecessary. The forbidden food list contained only food items with a manganese versus iron ratio of 0,33 or more. In case a participant wanted to eat a food item that was not included on the food list, they had to ask for permission. Since only foods items could be eaten from the recommended food list and neutral food list, it might seem odd that a forbidden food list was included. However, since the participants could already see the food items on the forbidden list, it prevented the participants from asking the researchers if these food items on the forbidden list might be allowed. The method of calculation of the food items is described in detail in the List of References in this invention. The manganese versus iron ratio of the food items was not supplied to any of the test participants.

Tables 1-3 list recommended, neutral and forbidden food items, respectively. The Tables display the list of food items referred to in the Examples below. The participants were only allowed to consume foods from the "recommended foods" and "neutral foods" list. In case a food item was not listed, the participants had to request the principal researcher if they could eat that non-listed item or not. A table of "forbidden foods" (Table 3) was included to inform participants upfront about food items which were not included in the "recommended foods" nor in the "neutral foods". This also prevented participants from asking permission to eat these particular food items. Also included in the tables are the manganese versus iron ratios of the various items on the food lists. The participants did not receive information on the manganese versus iron ratio and were not aware of this issue. Recommended foods were food items with a manganese versus iron ratio of less than 0,33. Forbidden foods were food items with a manganese versus iron ratio of 0,33 or more. Neutral foods did not include manganese.

Table 1: "Recommended foods'

Recommended foods Recommended foods (cont'nd)

Mn/Fe Mn/Fe ratio ratio

Anise 0,06 Ketchup 0,20

Apples 0,00 Kidney beans 0,12

Apricots 0,25 Leek 0,18

Asparagus 0,22 Lentils red / green 0,17

Avocado 0,17 Meat (all meats) < 0,32

Basil 0,08 Mint (peppermint, herb) 0,24

Bell Pepper 0,25 Mustard (prepared) 0,27

Black beans 0,22 Oatmeal 0,00

Broccoli 0,29 Oyster mushrooms 0,08

Brussels Sprouts 0,21 Oysters (mollusk) 0,12

Capers 0,06 Pasta (white) 0,23

Carrots 0,22 Pear 0,00

Cashews 0,25 Peas 0,32

Celery 0,17 Pepper (black pepper) 0,19

Chard 0,22 Potatoes (N B no sweet potatoes) 0,18

Cherries 0,25 Pumpkin (cooked) 0,17

Chili Pepper 0,17 Pumpkin seeds 0,20

Chive 0,25 Radicchio 0,17

Coriander leaves (raw) 0,22 Raisins 0,16

Coriander seeds 0,12 Rocket 0,20

Couscous 0,25 Rosemary 0,07

Cumin 0,05 Scallions, onions 0,13

Curry powder 0,15 Sesame seeds 0,17

Dark chocolate Shrimp 0,04

0,16

(preferably at least 70% cocoa) Soy Sauce 0,21

Dill 0,08 Spinach 0,27

Eggs 0,00 Sugarsnaps 0,10

Figs (preferably dried) 0,25 Thyme (dried) 0,06

Grapes 0,25 Tomatoes 0,20

Green Beans 0,20 Wheat flour (white) 0,15

Yellow corn cereal 0,19 Table 2: "Neutral foods'

Neutral foods

Mn/Fe ratio

All dairy products

(milk, cheese, goat's milk etc) 0

All oils 0

Carbonated beverages 0

Cheese 0

Coffee 0

Fish 0

Lemon juice 0

Mango 0

Mayonnaise 0

Melon (cantalon) 0

Olives 0

Orange juice 0

Tangerines 0

Water (still and carbonated) 0

Table 3: "Forbidden foods'

By only eating from the recommended and neutral food list (Tables 1 and 2), participants 1, 2 and 3 consistently ate only food items that contained at least three times more iron than manganese (since the manganese versus iron ratio of each food item was less than 0,33). With the food list,

participants 1, 2 and 3 worked (without knowing it) consistently towards obtaining or maintaining a lower manganese versus iron ratio than one within the blood sample (whereby the manganese is measured in nmol/1 and the iron concentration is measured in mol/l).

Participant 4 was asked to do nothing and eat as he normally did. He was the control group in this case.

General materials and methods

Manganese concentration versus iron ratio in a blood sample: The

manganese concentration versus iron ratio in a blood sample can be calculated by first measuring the manganese concentration in a blood sample (measured in nmol/1) and the iron concentration in a blood sample (measured in mol/l). Both measurements can be performed at a laboratory. The skilled person is well aware of suitable methods for measuring iron levels and manganese levels within a blood sample from a subject. The manganese amount in nmol/1 is then divided by the iron amount in mol/l to obtain the manganese versus iron ratio of a blood sample. For example: if the manganese concentration in a sample is 12 nmol/1 and the iron

concentration in a sample is 24 mol/l, the manganese concentration versus iron ratio in a blood sample in this example is 0,5 (12 nmol/1 divided by 24 mol/l).

The manganese concentration versus iron ratio in a macrophage:

The manganese concentration versus iron ratio in a macrophage can be calculated by first measuring the manganese concentration macrophages (measured in nmol/1) and the iron concentration in macrophages (measured in mol/l). Both measurements can be performed at a laboratory. The skilled person is well aware of suitable methods for measuring iron levels and manganese levels within microphages from a subject. The manganese amount in nmol/1 is then divided by the iron amount in mol/l to obtain the manganese versus iron ratio of a blood sample.

ELISA test performed on the blood samples of the participants in the Examples: An ELISA Enzygnost^© Lyme link VlsE IgG test from Siemens Healthcare Diagnostics GmbH, Eschborn, Germany was performed on the participants of the Examples. The ELISA Enzygnost^® Lyme link VlsE IgG test is based on a detergent extract from B. afzelii strain PKo, mixed with recombinant VlsE derived from B. afzelii, B. garinii and Borrelia burgdorferi s.s.. The test material can consists out of plasma or serum. The test was automatically processed and interpreted as recommended by the

manufacturer. Validation for this ELISA test can be found at (Ang et. al. 2011). The results is measured in kU/1. A negative result out of a sample consist out of a result lower than 4 kU/1, whereas a positive result out of a sample consist out of a result of 4 kU/1 or higher.

Western blot: The Western blot is a method of assaying for the presence of a particular protein within a sample. The Western blot test, also called immunoblot, seperates Borrelia burgdorferi proteins to their particle weights by using denaturing gel electrophoresis and then transferring these proteins electrophoretically (blotting) to a solid support matrix, which is generally but not limited to a nitrocellulose (NC), or polyvinylidene fluoride (PVDF) membrane. The transfer of the proteins is followed by

immunological detection of these proteins. The most common used Western blot tests for Lyme Disease can detect Borrelia burgdorferi IgM antibodies as well as Borrelia burgdorferi IgG antibodies.

The Western blot is used as a confirmation test after a positive or dubious result from an ELISA test or immunofluorescence assay for the detection of a Borrelia burgdorferi infection. The primary reason for performing the Western blot as a confirmation test for the detection of a Borrelia

burgdorferi infection is to make a distinction between the antibodies which are a cause of cross-reacting antibodies and antibodies caused by a

symptomatic or an asymptomatic infection of Borrelia burgdorferi. Not only the antigens used between Europe and the United States are different, also the criteria for interpreting the Western blot bands for Borrelia burgdorferi are different (Hauser, 1999). In Europe, multiple Borrelial subspecies can be the causal agent of Lyme disease, namely Borrelia burgdorferi s.s., Borrelia afzelii and Borrelia garinii. In Europe therefore, due to the occurrence of multiple Borrelial subspecies, an immunoblot with a native antigen of Borrelia garinii s.s. can cause the presence of Borrelia afzelii to be overlooked (Hauser, 1999; Kaiser, 2000). In Europe, immunoblots containing recombinant antigens result in the highest validity (Hauser, 1999). An advantage from recombinant antigens over native antigens, is the consistent quality of recombinant antigens in comparison to the inconsistent quality of native antigens in immunoblots which can vary from batch to batch (Goossens et.al., 1999). Another advantage of the recombinant-immunoblot over the native- immunoblot is that the recombinant-immunoblot is easy to interpret. The native-immunoblot however is difficult to interpret especially between the specific and cross-reactive immunobands and should always be interpreted by a skilled person.

In the United States however, Lyme disease is mainly caused by Borrelia burgdorferi s.s.. Consequently in the United States, an

immunoblot containing a native antigen of Borrelia burgdorferi s.s. will result in an optimal specificity I . Commercially recombinant -immunoblots can vary from company to company in sensitivity as well as specificity. The Centers for Disease Control and Prevention (CDC) in the United States provides guidelines for their proposed two-tier testing decision tree. The first step consist of a test serum by enzyme immunoassay (EIA) or an immunofluorescence Assay (IF A) or an equivalent method. The second step consists of using a highly specific Western blot to confirm positive or equivocal results from test of the first step. According to the CDC, an IgM Western blot is considered positive if any two out of the following bands are present: p24 (OspC), p39 (BmpA), p41 (Fla). An IgG Western blot is considered positive if any five out of the ten following bands are positive: l8, p21 (which is the same as p24 OspC), p28, p30, (BmpA), p41 (Fla), p45, p58 (not GroEL), p66, p93.

Example 1: Participant 1:

Demographics: Man between the age 40 and 45

Historic ELISA and Western blot test results testing for Borrelia burgdorferi (all taken at: Medische Laboratoria Dr. Stein & Colleg

Historic C6 ELISA test testing for antibodies against Borrelia burgdorferi from Reinier MDC, Medisch Diagnostisch Centrum:

NB this C6 ELISA from Reinier MDC, Medisch Diagnostisch Centrum considered positive in case the ratio is above 1,2. Previous antibiotics treatments included:

^• Minocycline in combination with plaquenil

^• Ciprofloxacine in combination with plaquenil

^• Doxycycline

· Azitromycine in combination with plaquenil

^• Metronidasol

On time point 1 (January 16^th 2015), blood samples were taken for the ELISA IgG test testing for antibodies oiBorrelia burgdorferi as well as for measuring the manganese and iron concentration in order to determine the manganese versus iron ratio in the blood sample. At that particular time point, the participant also received the dietary recommendations

represented by the food lists of Tables 1-3, which diet the participant was obliged to follow. On time point 2 (February 5^th 2015), blood samples were taken again for the ELISA IgG test testing for antibodies oiBorrelia burgdorferi as well as to measure the manganese and iron concentrations in order to again establish the manganese versus iron ratio in blood. The results from the participants were as follows:

Results example 1: Participant 1

Nb. The above ELISA test from Medische Laboratoria Dr Stein & Collegae is considered positive in case the result from the ELISA test is 4 kU/1 (four kU/1) or greater.

This result is in line with the invention. The manganese versus iron ratio has dropped from above 1,00 to less than 1,00 (from 1, 12 to 0,52) during the test and the ELISA test signal has dropped 38% accordingly .

The ELISA test was performed by using a Luminex 200™ device, the method involving coating microp articles with the recombinant Borrelia burgdorferi antigens OspA, OspC, p lOO, Vise, p39, p38 and p l8. After incubating the microp articles with blood serum of the subject, the particles were washed and incubated with antibodies against human IgG and/or IgM Again, a washing step was performed and fluorescence is determined as a measure for the amount of IgG or IgM.

Iron levels in serum were measured with the Cobas 6000® device (Roche Diagnostics, Switzerland), the method involving an absorption test wherein Fe³⁺ is converted in Fe²⁺. Fe²⁺ is allowed to react with ferrozine so as to produce light which was measured by spectroscopy. Output data was presented in units of mol/l.

Manganese levels in serum were measured with an Agilent ICP-MS 7500ce device (Agilent Technologies, US), the method involving inductively coupled plasma mass spectrometry (ICP-MS). Output data was presented was in units of nmol/1. Example 2: Participant 2:

Demographics: Woman between the age 35 and 40.

Historic ELISA and Western blot test results testing for Borrelia

burgdorferi (all taken at: Medische Laboratoria Dr. Stein & Colleg

NB this C6 ELISA from Reinier MDC, Medisch Diagnostisch Centrum is considered

positive in case the ratio is above 1,2. Previous antibiotics treatments included:

^• Azitromycine

^• Ciprofloxacine On time point 1 (January 16^th 2015), a blood sample was taken. At that particular date, the participant also received dietary recommendations represented by the food lists of Tables 1-3. Only food items from the recommended list and neutral list on the food list from Tables 1 and 2 could be eaten. The food items on the recommended list only had manganese versus iron ratios of less than 0,33. The forbidden food items had

manganese versus iron ratios of 0,33 or more. The participants only received the food list, not the manganese versus iron ratios of food items. On time point 2 (February 5^th 2015), a second blood sample was taken. On both blood samples an ELISA test testing for antibodies against Borrelia burgdorferi from Medische Laboratoria Dr Stein & Collegae was performed. Also on both blood samples, manganese levels and iron levels were measured by the laboratory Reinier Medisch Diagnostisch Centrum. The results were as follows:

Results example 2: Participant 2:

NB the above ELISA test from Medische Laboratoria Dr Stein & Collagae is considered positive in case the result from the ELISA test is 4 kU/1 (four kU/1) or greater.

This result is in line with the invention. The manganese versus iron ratio has dropped from above 1,00 to less than 1,00 (from 2,30 to 0,50) during the test and the ELISA test results have dropped 17% accordingly. The method and means for performing the ELISA test and measuring iron and manganese concentrations in serum are as indicated hereinabove.

Example 3: Participant 3:

Demographics: Woman between 45 and 50 years old.

Historic ELISA and Western blot test results testing for Borrelia burgdorferi (all taken at: Medische Laboratoria Dr. Stein & Collegae):

NB this C6 ELISA from Reinier MDC, Medisch Diagnostisch Centrum is considered positive in case the ratio is above 1,2.

Previous antibiotics treatments included:

^• Minocycline in combination with plaquenil

^• Clioquinol

· Hydroxyzine

On time point 1 (January 16^th 2015), a blood sample was taken. At that particular date, the participant also received dietary recommendations represented by the food lists of Tables 1-3. Only food items from the recommended list and neutral list on the food list from Tables 1 and 2 could be eaten. The food items on the recommended list only had manganese versus iron ratios of less than 0,33. The forbidden food items had

Results example 3: Participant 3:

This result is in line with the invention. It does not matter that the manganese versus iron ratio in the blood sample has increased from 0,48 to 0,63 as long as the foresaid ratio is less than one.

The manganese versus iron ratio at time point one as well as time point 2 is less than 1,00 and the ELISA test results have dropped 17% accordingly. It is important to note that as long as the manganese versus iron ratio is less than 1,00 the antibodies against Borrelia burgdorferi will decrease. It is interesting to note that the manganese amount of participant 3 has increased during the test while the ELISA IgG test result decreased. The Examples of the present invention indicate that maybe not manganese determines the virulence of Borrelia burgdorferi but a manganese versus iron ratio higher than one within a sample as described in the present invention may determine the virulence of Borrelia burgdorferi (and therefore the change in antibodies against Borrelia burgdorferi). This finding is in line with the.

The method and means for performing the ELISA test and measuring iron and manganese concentrations in serum are as indicated hereinabove.

Example 4 : Participant 4 (control group):

Demographics: Man between the age 55 and 60 years old.

Historic ELISA and Western blot test results testing for Borrelia

burgdorferi (all taken at: Medische Laboratoria Dr. Stein & Collegae, Maastricht, The Netherlands):

Historic C6 ELISA test testing for antibodies against Borrelia burgdorferi was performed by Reinier MDC, Medical Diagnostic Centre in Spijkenisse, Netherlands. The results (considered positive in case the ratio is above 1,2) were as follows

Previous antibiotic treatments included:

· Azitromycine in combination with plaquenil.

^• Minocycline in combination with plaquenil.

^• Zinnat in combination with plaquenil.

It is important to note, that this participant did not receive the dietary recommendations represented by the food lists of Tables 1-3. Therefore he did not consistently work towards maintaining a manganese versus iron ratio within the blood sample of less than one. His manganese versus iron ratio within the blood sample could have shifted below and above one at any time during the duration of the test.

On time point 1 (January 21^st 2015), blood samples for both tests were taken. On time point 2 (February 11^th 2015), blood samples were taken again. On both blood samples an ELISA test testing for antibodies against Borrelia burgdorferi from Medische Laboratoria Dr Stein & Collegae was performed. Also on both blood samples, manganese levels and iron levels were measured by the laboratory Reinier Medisch Diagnostisch Centrum. The results were as follows: Results example 4: Participant 4:

NB the above ELISA test from Medische Laboratoria Dr Stein & Collegae is considered positive in case the result from the ELISA test is 4 kU/1 (four kU/1) or greater.

This result is in line with the invention. The manganese versus iron ratio have increased from lower 0,61 at Jan 21^st 2015 to 1,73 at Feb 11^th 2015 and the ELISA test results have risen 5% accordingly (from 220 to 230).

NB This participant in example 4 was considered to be a control group and was not given the dietary recommendations represented by the food lists of Tables 1-3. Therefore it is important to note, that the

manganese versus iron ratio within the blood of participant 4 could have shifted below and above one at any time during the duration of the test. Due to this possible fluctuation of the manganese versus iron ratio within the blood, the ELISA IgG test result from time point 2 could have been lower, higher or stayed the same compared to the ELISA IgG test result from time point 1.

Due to this possible fluctuation of the manganese versus iron ratio within the blood of participant 4, it was predicted that the ELISA IgG test result from participant 4 would fluctuate less than the other three participants.

Summary examples:

No diet: indicates non compliance with the diet.

From participants 1, 2 and 3 the ELISA IgG test results significantly decreased during the test (respectively a 38% decrease, 17% decrease and 17% decrease). The structural eating pattern of eating solely food items with a manganese versus iron ratio of less than 0,33, resulted in a significant decrease in the ELISA IgG test results, which is in line with the invention.

The ELISA IgG test results of participant 4 is higher at time point 2 than at time point 1 and the manganese versus iron ratio in the blood has switched from below one to above one during the test (from a ratio of 0,61 to 1,73). This may seem in line with the invention but may as well be coincidental. Due to the randomness of the eating pattern of participant 4, any absolute change in ELISA IgG test result of participant 4 (as the control group) is unrelated to the invention. Altering the antibodies against

Borrelia burgdorferi in a sample may take more than a few days, while altering the manganese versus iron ratio from below one to above one (or vice versa) may take less time. Interestingly, the relative change in ELISA IgG test result from partici ant 4 (as the control group) compared to the other participants did not change nearly as much as the other participants. As mentioned before, the diet of participant 4 was random and as such it is likely that participant 4 both has eaten food items with a manganese versus iron ratio higher than 0,33 and lower than 0,33 at multiple times during the test. This in contrast with the other participants who structurally only ate food items with a manganese versus iron ratio lower than 0,33. Therefore the relative small change in ELISA IgG test result from participant 4 as the control group compared to the relative big changes in ELISA IgG test results from other participants, may be explained by the invention.

Example 5: Counteracting facial paralysis in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting facial paralysis are formed. The first patient group receives pill A by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, facial paralysis parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non- treated control patients, wherein when a is different from b, it is

demonstrated that pill A is effective in the treatment of facial paralysis in Lyme's disease patients. Facial paralysis parameters include closure of eyelids and drifting up of eye on affected side (Bell phenomenon).

Pill A comprises: (i) 2500 microgram of ferrous iron in the form of ferrous sulphate, (ii) 250 microgram of manganese in the form of manganese sulphate, (iii) 250 milligram of ascorbic acid, (iv) 750 milligram of

methylsulfonylmethane, (v) 200 milligram of magnesium citrate, (vi) 500 microgram of folic acid, (vii) 500 microgram of cobalamin, and (viii) 250 milligram of N-acetyl-cysteine (NAC).

Example 6: Counteracting dioplopia in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting dioplopia (double vision) are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, dioplopia parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the

treatment of dioplopia in Lyme's disease patients. Dioplopia parameters are generally assessed by answering questionnaires on vision.

Example 7: Counteracting tinnitus in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting tinnitus are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, tinnitus parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of tinnitus in Lyme's disease patients. Tinnitus parameters are generally assessed by audiological examination. Example 8: Counteracting unexplained weight loss in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting unexplained weight loss are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for four weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, unexplained weight loss parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of unexplained weight loss in Lyme's disease patients. Unexplained weight loss parameters are generally assessed measuring body weight. Example 9: Counteracting chest pain (angina) in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting chest pain are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, chest pain parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of chest pain in Lyme's disease patients. Chest pain parameters are assessed by a medical doctor.

Example 10: Counteracting arthritis in Lyme's disease patients with a composition of the invention. Two patient groups (each n=10) suffering from Lyme's disease and exhibiting arthritis are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, arthritis parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of arthritis in Lyme's disease patients. Arthritis parameters are assessed by a medical doctor.

Example 11: Counteracting sleep disturbances in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting sleep disturbances are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, sleep disturbance parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of sleep disturbances in Lyme's disease patients.

Example 12: Counteracting tingling in fingers in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting tingling in fingers are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, tingling in fingers is measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of tingling in fingers in Lyme's disease patients. Tingling in fingers is generally assessed by a medical doctor.

Example 13: Counteracting cognitive problems in Lyme's disease patients with a composition of the invention.

Two patient groups (each n=10) suffering from Lyme's disease and exhibiting cognitive problems are formed. The first patient group receives pill A (as indicated hereinbefore) by oral administration three times daily for two weeks, while the second patient group is non-treated or receives a placebo. Two weeks after treatment, cognitive problem parameters are measured in both patient groups, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, it is demonstrated that pill A is effective in the treatment of cognitive problems in Lyme's disease patients.

Example 14: Method for typing a sample for the presence or absence of antibodies against Borrelia burgdorferi .

Two patient groups (each n=10) infected with Borrelia burgdorferi are formed. Borrelia burgdorferi antibody titers are measured before treatment by ELISA in a serum sample of the patients. The first patient group receives pill B (Pill B comprises 35 mg manganese and 175 mg acetyls ahcy lie acid) by oral administration once a day for two weeks, while the second patient group is non-treated or receives a placebo. One week after treatment, antibodies against Borrelia burgdorferi in a serum sample of patients of both patient groups are measured by ELISA, wherein a is the outcome for treated patients, b is the outcome for non-treated control patients, wherein when a is different from b, in view of the pre-treatment control values, it is demonstrated that pill B increases Borrelia burgdorferi antibody titers and thereby provides for more reliable typing of a sample for the presence or absence of antibodies against Borrelia burgdorferi. REFERENCE LIST

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Grab, D. et al. (2013). A Manganese-rich Environment Supports Superoxide Dismutase Activity in a Lyme Disease Pathogen, Borrelia burgdorferi.

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http://users.rcn.eom/jkimball.ma.ultranet/BiologyPages/R/ROS.html Luger, S. (1990). Serologic tests for Lyme disease. Interlaboratory

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Nichols, T., Whitehouse, C, & Austin, F. (2000). Transcriptional analysis of a superoxide dismutase gene of Borrelia burgdorferi. FEMS Microbiology Letters, 183(1), 37-42. doi: 10.1111/j. l574-6968.2000.tb08930

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Claims

1. Method for typing a sample for the presence or absence of antibodies against Borrelia burgdorferi, comprising the steps of a) providing a sample, preferably a blood sample, of a subject infected, or suspected of being infected, with Borrelia burgdorferi; wherein in said subject the manganese concentration has been increased through

administering, preferably orally, manganese and/or wherein in said subject the iron concentration has been decreased through administering, preferably orally, an iron-sequestering compound, preferably acetyls alicy he acid; b) detecting antibodies, against Borrelia burgdorferi using an assay for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme- Linked Immuno Sorbent Assay (ELISA) or Western blot. c) typing said sample for the presence or absence of antibodies, against Borrelia burgdorferi.

2. Method according to claim 1, wherein in said subject the manganese concentration has been increased and/or wherein in said subject the iron concentration has been decreased so as to cause in the blood of said subject a ratio of manganese to iron higher than 1.

3. Method according to any one of claims 1-2, wherein said manganese and/or iron-sequestering compound are in a composition or a combination.

4. Method for increasing the sensitivity of an immunoassay for detecting Borrelia burgdorferi in a subject, the method comprising a) providing a sample, preferably a blood sample, from a subject infected, or suspected of being infected, with Borrelia burgdorferi; wherein in said subject the manganese concentration has been increased through

administering, preferably orally, manganese and/or wherein in said subject the iron concentration has been decreased through administering, preferably orally, an iron-sequestering compound, preferably acetyls alicy e acid; b) detecting antibodies against Borrelia burgdorferi using an assay for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme- Linked Immuno Sorbent Assay (ELISA) or Western blot.

5. The method according to claim 4, wherein in said subject the manganese concentration has been increased and/or wherein in said subject the iron concentration has been decreased so as to cause in the blood of said subject a ratio of manganese to iron higher than 1.

6. Use of manganese and/or an iron-sequestering compound, as a supplement in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method according to any one of claims 1-5.

7. Manganese and/or an iron-sequestering compound for use as a pharmaceutical compound in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method according to any one of claims 1-5.

8. A combination or supplement, preferably dietary or pharmaceutical, comprising manganese and/or an iron-sequestering compound, optionally further comprising one or more (pharmaceutically) acceptable excipients.

9. The combination or supplement according to claim 8, wherein the iron-sequestering compound is acetyls alicy lie acid.

10. The combination or supplement according to claim 8 or 9, wherein the combination or supplement, when administered to a subject, establishes in said subject, preferably in the blood of said subject, a ratio of manganese to iron higher than 1.

11. The combination or supplement according to claims 8- 10, wherein the combination is for use in the detection of Lyme's disease, preferably said detection of Lyme's disease involving performance of a method according to any one of claims 1-5.

12. A kit of parts comprising manganese and an iron-sequestering compound, said manganese and iron-sequestering compound optionally formulated with one or more (pharmaceutically) acceptable excipients, preferably for use in detecting Lyme's disease.

13. The kit of parts of claim 12, wherein said detection of Lyme's disease involves performing a method according to any one of claims 1-5.

14. A combination or supplement, preferably dietary or pharmaceutical, comprising iron and optionally manganese, the combination, supplement or composition optionally further comprising one or more (pharmaceutically) acceptable excipients.

15. The combination or supplement of claim 14, comprising 50-30000 microgram of iron, preferably ferrous iron.

16. The combination or supplement of claim 14 or claim 15, further comprising 1-3000 microgram of manganese, wherein the weight ratio of iron to manganese, measured in the same unit of weight, is at least 3: 1.

17. The combination or supplement of claims 14- 16, further comprising one or more compounds selected from the group formed by, or consisting of, ascorbic acid, methylsulfonylmethane (MSM), magnesium, folic acid, vitamin B 12 (cobalamin) and/or cysteine.

18. The combination or supplement of claims 14- 17 for use as a

medicament.

19. The combination or supplement of claims 14- 18 for use in

counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

20. Iron, preferably ferrous iron, for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject.

21. The combination or supplement for use of claim 19, wherein the combination or supplement establishes, when administered to said subject, in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1.

22. A container for holding a combination or supplement according to any one of claims 14- 17, the combination or supplement in the container having an overall iron to manganese weight ratio of at least 3: 1.

23. A kit of parts comprising at least two foodstuffs, preferably different foodstuffs, for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, wherein the foodstuffs comprised in the kit have an overall iron to manganese weight ratio of at least 3: 1.

24. The kit of parts according to claim 23, wherein the at least two foodstuffs, when administered to said subject, establish in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1.

25. The kit of parts according to claim 23 or 24, wherein the at least two foodstuffs are selected from the group formed by, or consisting of, anise, apples, apricots, asparagus, avocado, basil, bell pepper, black beans, black pepper, broccoli, brussels sprouts, capers, carrots, cashews, celery, chard, cherries, chili pepper, chive, coriander leaves (raw), coriander seeds, couscous, cumin, curry powder, dark chocolate (preferably at least 70% cocoa) , dill, eggs, figs (preferably dried), grapes, green beans, ketchup, kidney beans, leek, lentils (red or green), meat (all meats), mint

(peppermint, herb), mustard (prepared), oatmeal, oyster mushrooms, oysters (mollusk), pasta (white), pear, peas, potatoes (nb no sweet potatoes), pumpkin (cooked), pumpkin seeds, radicchio, raisins, rocket, rosemary, scallions, onions, sesame seeds, shrimp, soy sauce, spinach, sugarsnaps, thyme dried), tomatoes, wheat flour (white), yellow corn cereal.

26. A foodstuff for use in counteracting, controlling or reducing the symptoms of Lyme's disease in a subject, wherein said foodstuff, when administered to said subject, establishes in said subject, preferably in the blood of said subject, a ratio of manganese to iron lower than 1.

27. A method of controlling or modulating the immune response against Borrelia burgdorferi in a subject infected, or suspected of being infected, with Borrelia burgdorferi, comprising the step of

a) administering a combination or supplement according to claims 8- 10, or a combination or supplement according to claims 14- 17.

28. The method according to claim 27, wherein the combination or supplement according to claims 8- 10 is administered in a Borrelia

burgdorferi antibody-titer increasing amount, or wherein the combination or supplement according to claims 14- 17 is administered in a Borrelia burgdorferi antibody-titer reducing amount.