NL2014614B1

NL2014614B1 - Method and means for increasing antibodies against Borrelia burgdorferi and increasing antigen of Borrelia burgdorferi.

Info

Publication number: NL2014614B1
Application number: NL2014614A
Authority: NL
Inventors: Timmermans Dennis
Original assignee: Pld Biochemistry B V
Priority date: 2015-04-10
Filing date: 2015-04-10
Publication date: 2017-01-25
Also published as: WO2016163888A1; 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.

Description

Titel: 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.

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 laboratory method for the diagnosis of Lyme disease. However, the insensitivity of the assays is a frequent problem (Luger, 1990). The test can give false negatives when the concentration of antibodies is too low or when the antibodies do not react well with the commercially produced antigens and false positives if the person has antibodies for a similar antigen non-related to Lyme Disease. The RIVM (“Rijksinstituut voor Volksgezondheid en Milieuhygiëne”, the Dutch government institution for Health and Environmental hygiene) and the VUMC (“Vrije Universiteit Medisch Centrum”, the medical center from the Dutch university “Vrije Universiteit”) conducted a research in 2011 to estimate the sensitivity of the ELISA test. The research estimated that the sensitivity of the ELISA test, performed by seven different laboratories on the patients highly suspected with Borrelia burgdorferi, lies between 34% and 59% whereby 36% of the Borrelia burgdorferi infections in suspected patients were missed altogether. The current low sensitivity of the diagnostic tests for Lyme disease highlight the need for means and methods that either increase the concentration of antibodies against Borrelia burgdorferi or increase the quality of commercially produced antigens as to increase the overall sensitivity of the immunoassays detecting Lyme disease. The present invention is directed to fulfilling the need of increasing the concentration of antibodies against Borrelia burgdorferi.

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 to be 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. 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 acetylsalicylic 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 beheved 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.

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 sample, preferably a blood sample, of a subject infected, or suspected to be 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 acetylsalicyhe 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 to be 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 acetylsaheylie 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 composition according to the invention, the iron-sequestering compound is acetylsalicylic acid.

The Examples of the present invention show that within a sample, a manganese (measured in nmol/1) versus iron ratio (measured in pmol/l) lower than one or higher than one is decisive for the decrease respectively increase of the 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 “acetylsalicylie acid”, as used herein, refers to the acetylated derivative of salicylic acid; also known as aspirin (TheFreeDictionary.com (1), 2015). The structural formula of acetylsalicylic 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 is preferred.

The term “antibody” , as used herein, refers to a mammalian, 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 immunoglobulin G (IgG) and/or immunoglobulin M (IgM), present in a sample, preferably blood sample, of a mammalian, 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 oiBorrelia burgdorferi.

The term “antigen”, as used herein, refers to a molecule capable of generating an immune response, preferably an antibody response, in a mammalian, 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 afzelii, 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 acetylsalicylic 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 of Borrelia burgdorferi. Detecting of Borrelia 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 Borrelm 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 “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+

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

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 (O2-) 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 blood sample of a subject. 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 manganese and/or an iron-sequestering compound such as acetylsalicylic acid.

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 Borreba burgdorferi in a subject. If said antibodies are present, said subject is typed as being infected with Borreba burgdorferi.

The term “Western blot”, as used herein, refers to the Western blot test, also cabed immunoblot, which separates proteins to their particle weights by using denaturing gel electrophoresis and then transferring these proteins electrophoreticaby (blotting) to a sobd support matrix, which is generaby a nitrocebulose (NC), or polyvinyhdene fluoride (PVDF) membrane. The transfer of the proteins is fobowed by immunological detection of these proteins. For Lyme disease, the Western blot preferably detects IgM antibodies against Borrelia burgdorferi as web as IgG antibodies against Borrelia burgdorferi. A skibed person knows that different laboratories can use different antigens for detecting antibodies against Borrelia burgdorferi. The skibed 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.

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 cytosobc 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 (ScienceDaby, 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 limitation 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 (O2-). 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 (O2-) into either ordinary molecular oxygen (O2) or hydrogen peroxide (H202). 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 andMnSOD 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 dehvered via embodiments but not hmited 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 hmited to, oral, rectal or parenteral administration. Oral administration offers advantages over other routes of administration.

In a preferred embodiment, pihs represent one form of a composition of this invention comprising the inventive combination of manganese and acetylsalicylie acid. When in the form of a single dosage unit, such as a pill, the composition may comprise about 175 mg acetylsalicylic acid in combination with about 35 mg manganese for instance acetylsalicylic 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 acetylsalicylic acid and for instance manganese preferable chelated manganese in the range 1 mg, 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 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.

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 hmited 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 ELISA test is based but not limited to on a (Ce) synthetic peptide that reproduces the sequence of a 26-mer invariable region of the surface antigen VlsE of Borrelia burgdorferi. 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: pl8, p22 OspC (which is the same as p24 OspC), p31 (OspA), p39 (BmpA), p58, p66 and p 100. • IgG antibodies against Borrelia burgdorferi: pl8, 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.: pl8, p21 OspC (which is the same as p22 OspC andp24 OspC), p28, p30, (BmpA), p41 (Flaggelin), p45, p58 (not GroEL), p66, p93.

It should be readily apparent to one of ordinary skill in the art that numerous modifications, alternative substitutions, alternative compositions, may be devised to the invention disclosed herein without departing from scope and spirit of the invention. The present invention is not limited by the selection of the particular assay format and is believed to encompass assay formats which are known to those of ordinary skill in the art. The present invention is not limited to the specific embodiments described herein which are intented as illustrations of various aspects.

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 pmol/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 pmol/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 pmol/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 acetylsalicylic acid is used. The skilled person will understand that acetylsalicylic acid is an anti-inflammatory agent against almost all pathogenic bacteria but not against Borrelia burgdorferi. The skilled person understands that acetylsalicylic 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 acetylsalicylic acid is not an anti-inflammatory 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 acetylsalicylie 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 to be infected with Borrelia burgdorferi, the method comprising the step: a) administering manganese and/or administering an iron sequestering compound, preferably acetylsalicylic acid, to said subject.

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 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 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 hsts of Tables 1-3, from which they were only allowed to eat from the recommended list and neutral hst. What the participants did not know was that each food item on the recommended food hst 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 hst 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 hst, they had to ask for permission. Since only foods items could be eaten from the recommended food hst and neutral food hst, it might seem odd that a forbidden food hst was included. However, since the participants could already see the food items on the forbidden hst, it prevented the participants from asking the researchers if these food items on the forbidden hst 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 hst of food items referred to in the Examples below. The participants were only allowed to consume foods from the “recommended foods” and “neutral foods” hst. In case a food item was not listed, the participants had to request the principal researcher if they could eat that non-hsted 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”

Table 2: “Neutral foods”

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 pmol/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 pmol/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 pmol/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 pmol/l, the manganese concentration versus iron ratio in a blood sample in this example is 0,5 (12 nmol/1 divided by 24 pmol/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 pmol/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 pmol/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 (IFA) 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: pl8, 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 & Collegae):

Historie 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 is 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 16th 2015), blood samples were taken for the ELISA IgG test testing for antibodies of Borrelia 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 5th 2015), blood samples were taken again for the ELISA IgG test testing for antibodies of Borrelia 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 .

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 & CoUegae):

Previous antibiotics treatments included: • Azitromycine • Ciprofloxacine

On time point 1 (January 16th 2015), a blood sample was taken. At that particular date, the participant also received dietary recommendations represented by the food hsts of Tables 1-3. Only food items from the recommended hst and neutral hst on the food hst from Tables 1 and 2 could be eaten. The food items on the recommended hst 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 hst, not the manganese versus iron ratios of food items. On time point 2 (February 5th 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.

Example 3: Participant 3:

Demographics: Woman between 45 and 50 years old.

Previous antibiotics treatments included: • Minocycline in combination with plaquenil • Clioquinol • Hydroxyzine

On time point 1 (January 16th 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 hst on the food list from Tables 1 and 2 could be eaten. The food items on the recommended hst 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 hst, not the manganese versus iron ratios of food items. On time point 2 (February 5th 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 & CoUegae 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 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.

Example 4 : Participant 4 (control group):

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

Historie 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 21st 2015), blood samples for both tests were taken. On time point 2 (February 11th 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 21st 2015 to 1,73 at Feb 11th 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:

Diet: Indicates compliance with the diet represented by the food list Fig. 1.; 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 fine 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 participant 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.

REFERENCE LIST

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Claims

A method for typing a sample for the presence or absence of anti-Borrelia burgdorferi antibodies, comprising the steps of: a) providing a sample of a patient infected or suspected of being infected with Borrelia burgdorferi; b) determining a manganese to iron ratio in the sample; c) detecting antibodies against Borrelia burgdorferi using a test for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme Linked Immuno Sorbent Assay (ELISA) or Western blot test, in the ratio as determined under step b) is greater than 1; and d) typing the sample for the presence or absence of antibodies against Borrelia burgdorferi based on the result of the test obtained under step c).

Method according to claim 1, wherein, if the ratio in step b) mentioned in said sample is equal to or lower than 1, said method further comprises the steps of: e) administering, preferably orally administering, a manganese and / or iron sequestering compound, preferably acetylsalicylic acid, to the patient; and f) optionally, determining a manganese to iron ratio in the sample; wherein steps e) and f) are performed after step b) and before step c).

A method for typing a sample for the presence or absence of anti-Borrelia burgdorferi antibodies, comprising the steps of a) providing a sample, preferably a blood or serum sample of a patient infected or suspected of being infected with Borrelia burgdorferi; wherein in the subject the manganese concentration is increased by administering, preferably orally, manganese and / or wherein in the subject the iron concentration has decreased by administering, preferably orally, an iron sequestering compound, preferably acetylsalicylic acid; b) detecting antibodies against Borrelia burgdorferi use of a test for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme - Linked Immuno Sorbent Assay (ELISA) or Western blot. c) typing the sample for the presence or absence of antibodies against Borrelia burgdorferi based on the result of the test obtained under step c).

The method of claim 3, wherein in said patient the manganese concentration is increased and / or wherein in said patient the iron concentration is lowered to result in a manganese to iron ratio in the blood or serum of said patient of greater than 1 .

The method according to any of claims 2-4, wherein the manganese and / or iron sequester compound is in a composition.

A method for increasing the sensitivity of an immunoassay to detect Borrelia burgdorferi in a patient, the method comprising a) providing a sample, preferably a blood or serum sample from a patient infected or suspected of being infected with Borrelia burgdorferi; wherein in the patient the manganese concentration is increased by administering, preferably orally, manganese and / or wherein in the patient the iron concentration has decreased by administering, preferably orally, an iron sequestering compound, preferably acetylsalicylic acid; b) detecting antibodies against Borrelia burgdorferi use of a test for the detection of antibodies against Borrelia burgdorferi, preferably an Enzyme-Linked Immuno Sorbent Assay (ELISA) or Western blot.

The method of claim 6, wherein the manganese concentration in the patient is increased and / or wherein the iron concentration in the patient is lowered to result in a manganese to iron ratio in the blood or serum of said patient greater than 1.

Use of manganese and / or an iron sequestering compound as a dietary supplement in the detection of Lyme disease, preferably wherein said detection of Lyme disease comprises the implementation of a method according to any of claims 2-7.

A manganese and / or an iron sequestering compound for use as a pharmaceutical compound in the detection of Lyme disease, preferably wherein said detection of Lyme disease comprises the implementation of a method according to any of claims 2-7.

A diet combination or pharmaceutical combination comprising manganese and an iron sequestering compound, optionally further comprising an (pharmaceutically) permitted excipient.

The composition of claim 9, wherein the iron sequestering compound is acetylsalicylic acid.