WO1996025155A1

WO1996025155A1 - Diagnosis of and compositions and methods for the treatment of disease

Info

Publication number: WO1996025155A1
Application number: PCT/IB1996/000222
Authority: WO
Inventors: Timothy Kilgour Roberts; Neil Roland Mcgregor; Henry Lawrence Butt; Richard Hugh Dunstan; Mariann Zerbes; Iven John Klineberg
Original assignee: Bioscreen Pty. Limited
Priority date: 1995-02-15
Filing date: 1996-02-15
Publication date: 1996-08-22
Also published as: AU4841296A; AUPN115095A0

Abstract

A variety of microbes, bacterial products and by-products and their association with chronic disease states are disclosed. These associations can be used to diagnose and effect proper treatment for chronic disease. In particular bodily fluids are analyzed from patients with chronic disease and the relative measurements of bacterial products and by-products are evaluated. In one embodiment, the bodily fluid is urine and the method for analysis is GC-MS. Also provided are methods for the treatment of chronic disease states including the generation of antibodies to selected products and by-products.

Description

DESCRIPTION

DIAGNOSIS OP AND COMPOSITIONS AMD METHODS PQR THE TREATMENT OP DISEASE

BACKGROUND OP THE INVENTION

I. Field of the Invention The present invention provides methods and

composition for the diagnosis and treatment of chronic disease states, for example, chronic pain and chronic fatigue. In particular, the association of certain microbes with chronic disease states provides targets for both diagnostic applications and therapeutic

intervention. Diagnosis of Chronic Fatigue Syndrome (CFS) is a particular embodiment of the invention.

II. Description of the Related Art

Chronic fatigue and chronic pain disorders encompass a wide range of varied and often ill-defined long-term pathological symptoms. Because of the broad range of these symptoms and the lack of consistent etiological agents, these diseases have proved difficult to define and no objective diagnostic tests currently are

available. Often, the various symptoms of chronic fatigue and pain are attributed to other pathological states or with psychological aberrations, but without any scientific foundation. This is understandable when the symptoms are listed: muscle pain, headache, nausea, stiffness, bloating, chest pain, back pain, fever, heart palpitations, sleep loss, memory loss, concentration loss, hostility, numbness, tingling, lymph node

tenderness, breathlessness and a host of other physical and mental problems. One of the more prominent chronic diseases is chronic fatigue syndrome (CFS). CFS is a debilitating multisystem illness that has been associated with viral infection, particularly infection with Epstein Barr

Virus. It is characterized by debilitating fatigue, cognitive disturbances, myalgia, headache, recurrent sore throat, low grade fever, gastrointestinal and urogenital symptoms and lymphodynia. CFS is currently diagnosed by compliance with (i) clinical definition, (ii) and

exclusion of other known fatigue-related diseases.

(Holmes et al . , 1988; Sharpe et al . , 1991; Lloyd et al . , 1990; Fukuda et al . , 1994). No validated diagnostic tests are available, however, and the lack of objective biochemical and/or cellular diagnostic markers has resulted in considerable confusion in diagnosing the syndrome and hampered the scientific investigation of its etiology and pathophysiology.

SUMMARY OP THE INVENTION

There is provided, in accordance with the present invention, methods for diagnosing a chronic disease state comprising (a) obtaining a sample from an animal

suspected of having a chronic disease state; and (b) identifying at least one microbial organism that is present in an amount that varies from the amount observed in a healthy animal. The method of identifying a microbe may be by microbial culturing or measuring a bacterial product or byproduct in said sample. The sample may be

cerebrospinal fluid, blood, urine, sputum, tears, sweat, feces, tissue or other suitable sample. It also may be desirable to perform the diagnosis in conjunction with performing of a patient survey. Further, the method may involve performing

chromatographic separation of said sample or, more specifically, determining the area under the peaks of a gas chromatograph profile of a urine sample. Relevant urine gas chromatograph markers include CFSUM1, CFSUM2, UM13, UM13A, UM14, UMS15, UM15A, UM15B, UM17 and UM28. Other bacterial products or by-products are

staphylococcal α, ß, γ, δ , δ-like and horse toxins and ß alanine.

The chronic disease state may be selected from the group consisting of chronic fatigue, chronic pain, chronic inflammation, chronic depression, tinnitus, heart palpitations, migraine, short term memory loss and vision disturbances. Chronic pain can be muscle pain, headache, joint pain, lymph node pain, back pain and chest pain.

Another aspect of the present invention involves the treatment of chronic disease states. For example, a method of treating a chronic disease state comprises (a) identifying an animal suspected of having a chronic disease state; and (b) providing to said animal at least one antibiotic. The antibiotic may be one that acts on bacterial ribosomes and/or that acts on bacterial DNA. Another method of treatment includes identifying an animal suspected of having a chronic disease state; and (b) providing to said animal at least one toxin binding protein. Still further, the method may include (a) identifying an animal suspected of having a chronic disease state; and (b) providing to said animal at least one nutritional supplement. These methods may be used separately or in conjunction with each other.

The methods include treatment and diagnosis of organisms selected from the group consisting of

Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. , Pseudomonas spp. , Bacillus spp. , Lister ia spp. ,

Enterobacteriacaceae spp. , Spirochaetes spp. and the cell wall deficient variants thereof. Where the organism is a Staphylococcus spp. , it may be S. warneri , S.

haemolyticus, S. xylosis, S. hominis, S. epidermidis and S. lugdunensis .

Another embodiment of the present invention is a purified antigen composition comprising (a) a lipid soluble exoprotein produced by a microbial organism selected from the group consisting of Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. , Pseudomonas spp. , Bacillus spp. , Listeria spp. , Enterobacteriaceae spp. , Spirochaetes spp. and the cell wall deficient variants thereof; and (b) a pharmaceutically acceptable carrier, buffer or diluent. Also, the purified antigen

composition may comprising (a) an antigen selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14, UMS15, UM15A, UM15B, UM17 and UM28; and (b) a pharmaceutically acceptable carrier, buffer or diluent. Yet another embodiment includes an antibody that recognizes an antigen selected from the group consisting Of CFSUM1, CFSUM2, UM13, UM13A, UM14 , UMS15, UM15A,

UM15B, UM17 and UM28. Also, the antibody may recognize a lipid soluble exoprotein produced by a microbial organism selected from the group consisting of Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. , Pseudomonas spp. , Bacillus spp. , Listeria spp. , Enterobacteriaceae spp. , Spirochaetes spp. and the cell wall deficient variants thereof. More specifically, the antibody may be to an exoprotein that is a staphylococcal α, ß, γ, δ, δ-like or horse toxin.

Yet still another embodiment is a method of

generating an immune response to a chronic disease state- associated compound comprising providing to an animal an antigen composition comprising (a) a bacterial product or by-product; and (b) a pharmaceutically acceptable

carrier, buffer or diluent. The bacterial product may be a toxin or an outer membrane protein and the bacterial by-product is CFSUM1, CFSUM2, UM13, UM13A, UM14, UM15, UM15A, UM15B, UM17, UM28 or ß alanine.

Yet still another embodiment involves a method of preventing a chronic disease state comprising providing to an animal an antigen composition comprising (a) a bacterial product or by-product; and (b) a

pharmaceutically acceptable carrier, buffer or diluent. The bacterial product may be a toxin or an outer membrane protein and the bacterial by-product is selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14, UM15, UM15A, UM15B, UM17, UM28 or ß alanine.

BRIEF DESCRIPTION OP THE DRAWING

FIG. 1 shows the total ion chromatogram of a urine sample from a chronic disease state patient. The

corresponding peak numbers, identity, retention time, retention indices and mean composition values for the twenty eight peaks are summarized in Table 1.

FIG. 2 shows a diagrammatic representation of the interrelationship of CFS symptoms and markers and the effect on the severity of the condition. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that certain microbial organisms are associated with the development of chronic disease states. As a result of this correlation, it now is possible, for the first time, to accurately diagnose a number of chronic fatigue and pain diseases. In addition, the availability of proven anti-microbial agents such as antibiotics and anti-microbial antibodies makes possible the effective treatment of a variety of chronic disease states. The invention is described in detail below.

I. Chronic Disease States

For the purposes of the present invention, a

"chronic disease state" will be defined as a pathologic condition that results from (i) the colonization of an animal by one or more microbes that are not normally part of the microbial flora of that animal, (ii) the absence of colonization of an animal by one or more microbes that are normally part of the microbial flora of that animal or (iii) an abnormally high or low level of colonization of an animal by one or more microbes when compared to the normal level of colonization of that animal by the microbe(s) . These different definitions may overlap in the sense that an animal may exhibit more than one of these phenomena at the same time. Colonization is distinguished from an infection in that an infection normally induces an inflammation in the host organism. Colonization, on the other hand,

typically does not result in an inflammation. Another distinguishing characteristic is the immune response of the host organism. Against an infectious organism, the healthy host's natural defenses will mount a response to the invading microbe; a colonization usually does not elicit an immune response from its host.

Currently, it is believed that the balance of commensal microbes is important to the health of the host animal. When certain non-commensal organism colonize the animal, toxins may be produced or the metabolites of the non-commensals may act as toxins, both causing

significant clinical effects.

In addition, non-commensals microbes may affect the growth of the commensal organism and alter the microbial balance. Alternatively, commensals may be reduced by some other biological or environmental factor. Because commensals can aid the host animal, their absence or diminution also can cause detrimental effects.

Another variation is where a commensal microbe is found in amounts far in excess of that seen in the healthy animal. Here, the commensal's metabolites, tolerated well at normal levels, are toxic when produced in larger amounts. This sort of "commensal" has, in fact, become "non-commensal" in the sense that it does not exist in harmonious coexistence with its host.

One of the problems previously associated with accurately diagnosing and classifying chronic diseases derived, in part, from the fact that so many chronic disease symptoms arise from other disease states. Thus, because of this inability to find a common causative agent for these diverse symptoms, these symptoms were either erroneously attributed to non-chronic diseases or erroneously assumed to be the result of psychological disturbances. One of the important aspects of the present invention is the ability to identify which of the chronic disease symptoms are truly indicative of the "chronic disease state" and, hence, treatable by various therapeutic regimens discussed below.

The following list of symptoms that arise from chronic disease is not meant to be exhaustive of such symptoms, nor is it reflective of those seen in every (or even most) chronic disease sufferers. Rather, this list merely is representative of such chronic disease

symptoms. Chronic disease state symptoms include

fatigue, chronic pain, chronic muscle pain, headaches, tension headaches, nausea, morning joint stiffness, urinary frequency, cervical node tenderness,

hyperaesthesia, allergies, palpitations, abdominal bloating, recurrent feverishness, cardiac dysrhythmias, axial node tenderness, recurrent sore throats, tinnitus, irritable bowel syndrome, dermatitis, low grade diarrhea, paraesthesia, groin node tenderness, bruxism, chest pain, constipation, sciatica, aphthous ulceration, dysuria, muscle soreness, trouble concentrating, trouble

remembering, weak feelings in body, restless sleep, back pain, upper back pain, lower back pain, trouble falling asleep, low energy, mind going blank, heavy limbs, loss of libido, faintness or dizziness, feeling blue,

numbness/tingling, slow for correctness, something wrong in body, decisions difficult, difficult awakening in morning, hot and cold spells, heart pounding/racing, no interest in things, breathlessness, trembling, chest pain, lump in throat, avoiding activity, somatization, obsessive compulsive behavior, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, psychoticism, widespread pain and regional pain.

It also has been observed that certain of these symptoms are associated with particular microbial

colonizations, i.e., that certain microbes are associated with particular symptoms. Thus, where particular symptoms are seen, it may prove instructive to look for particular organisms. Conversely, where certain microbes have been identified as having colonized a host, the attribution of symptoms to the presence of these microbes can serve to confirm the diagnosis of chronic disease and, hence, give more accurate information with regard to the particular treatment regimen to be employed.

Of particular interest is the disease referred to as Chronic Fatigue Syndrome (CFS) . Other variations of chronic fatigue disorders are Post-viral Fatigue Syndrome (CFS), Myalgic Encephalomyelitis (ME), Chronic Fatigue Immunodeficient Syndrome (CFIDS), Epidemic or Endemic Neurasthenia (EN), Gulf War Syndrome (GWS), Multiple Chemical Sensitivities (MCS) and Irritable Bowel Syndrome (IBS). Chronic pain, another major chronic disease state, exists in numerous variations. These include, but are not limited to, Fibromyalgia, Temporomandibular Joint Dysfunction (TMJ), Myofacial Pain Syndrome (MPS) and Repetitive Strain Injury (RSI).

II. Diagnosing Chronic Disease states

One embodiment of the present invention involves the diagnosis of chronic disease states by identifying the presence, absence, relative amount or combination of certain microbes in an animal. It is believed that information on any one of a number of different organisms will be instructive on the clinical state of the animal being diagnosed.

As stated above, chronic disease may be indicated by the mere presence or absence of a particular microbe, or it may involve a more subtle situation where the level of colonization of a host by a particular microbe is the causative factor. The determination may be made by looking directly for the presence of microbes or indirectly for the presence or absence of microbial products or by-products. This aspect of the invention is described in greater detail below. A. Microbes

The present invention contemplates the association of certain microbes with chronic disease states. For example, bacteria of the following species are considered to be agents of chronic disease: Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. , Pseudomonas spp. ,

Bacillus spp. , Listeria spp. , Enterobacteriacaceae spp. and Spirochetes spp. In a particular embodiment, the bacterial species is S. warneri , S. haemolyticus, S.

xylosis, S. hominis, S. epidermidis or S. lugdunensis.

In addition, important variations of the foregoing are the cell wall deficient variants thereof. In

response to external agents, certain microbes are able to dispose of their cell walls while continuing to grow.

These co-called "L-forms" often result from exposure of the microbes to anti-microbial agents or from other environmental stresses. These organisms become

particularly difficult to detect and treat when in the L-form. They may become "dormant," making therapies directed against microbial metabolism especially

ineffective, but later revert to the normal cell wall-containing form allowing for the recurrence of disease.

The culture conditions and the media requirements for each of these microbes are well known to those of skill in the art (Balows et al . , 1991). B . Markers

The present invention allows for the determination of a chronic disease state by direct analysis to measure the microbes, or by indirect analysis for the measurement of microbial products or by-products. A wide range of sample material may be employed for the analysis. For example, samples of bodily fluids including, but not limited to, cerebrospinal fluid, blood, urine, sputum, tears, sweat, feces or tissue may be analyzed. Tissue samples including scrapings and biopsies also are

envisioned.

As stated above, the analysis can involve

determining the presence, absence or amount of a microbe, or of microbial products or by-products. For the

purposes of this application, microbial products are those compounds synthesized and/or excreted by microbes. Examples would be microbially synthesized proteins or lipids, or the waste products of microbial metabolism.

Microbial by-products are those compounds generated as a result of microbial colonization but that are neither synthesized nor excreted by the microbe. These would include host products, and possibly the products of other microbes, that are generated in response to the chronic disease-associated microbe.

An example of a microbial product is a structural or secreted protein. Microbial toxins such as

staphylococcal a, β, γ, δ , δ-like toxins and horse hemolysin toxin are preferred embodiments. Other

microbial products are CFSUM1, CFSUM2, UM13, UM13A, UM14, UM15, UM15A, UM15B, UM17 and UM28. These products were identified by examination of urine samples by gas

chromatography/mass spectrometry (GG-MS) . In a

particular embodiment the bodily fluid analyzed is urine and the method of analysis is GC-MS. Table 1 identifies the 28 peaks analyzed thus far from urine samples. The conditions for collection and analysis of these samples are given in Example 2 and a representative total ion chromatograph of a urine sample from a patient with a chronic disease state is shown in FIG. 1. one of skill in the art would recognize that conditions for GC analysis could be modified, especially to affect longer or shorter retention times, without altering the diagnostic potential of the technique for CFS.

The appraisal of the symptom expression in chronic disease and healthy control subjects reveals an extensive range of symptoms associated with the chronic disease patients. The relative abundance of CFSUM1 is an

important factor in the urine profile discriminating between those subjects reporting and not reporting severe fatigue as discussed in Example 3. Many other symptoms, such as urinary frequency, nausea, dizziness, diarrhoea, muscle twitching, allergies, breathlessness and hyperaesthesia have been previously documented in CFS patients (Hyde et al . , 1992) but are not in the

definitions (Holmes et al . , 1988; Sharpe et al . , 1991; Lloyd et al . , 1990; Fukuda et al . , 1994). In addition to severe fatigue, the relative abundance of CFSUM1 is an important discriminant parameter for the expression of infection-related symptoms and musculoskeletal symptoms. The relative abundance of β-alanine is an important discriminant parameter for the expression of

gastrointestinal and genitourinary symptoms. These data establish a strong relationship between CFSUM1 and β-alanine with CFS and its core symptoms, and suggest that other previously documented symptoms not included in the definition are also discriminant for chronic disease. The relative abundances of CFSUM1 and β-alanine are also important factors correlated with the index. The

relative abundance of CFSUM2 is a primary correlate

(negative) for the neurological index and the VAPS. The present invention also correlates anomalies in a series of energy (TCA cycle and nitrogen transport) and neurotransmitter-related amino acid to a novel metabolite (chronic fatigue symptom urinary marker 1 or CFSUM1) .

Patients in a chronic disease state exhibit increases in five metabolites (aconitic and succinic acids, tyrosine, β-alanine, CFSUM1) and reductions in three metabolites (glutamic acid, alanine, unknown CFSUM2). No significant differences in the protein turnover markers, leucine and 3-methyl-histidine, were found which does not support an overt cytokine-induced condition. Chronic disease state patients have alterations in urinary metabolites that reflect anomalies in nitrogen and energy metabolism suggestive of a chronic low grade catabolic disturbance. CFSUM1, β-alanine and CFSUM2 were found by discriminant function analysis to be the 1st (p<0.00001), 2nd and 3rd discriminant variables for CFS. CFSUM1 and CFSUM2

correlated significantly with changes in TCA cycle intermediates and the nitrogen metabolism and

neurotransmitter amino acids whilst β-alanine correlated with changes in the urea cycle amino acids, and alanine and leucine.

The structure of CFSUM1 has been determined as amino-hydroxy-N-methyl-pyrrolidine. CFSUM1 has a great similarity with the neuroactive drug

3-amino-1-hydroxy-pyrrolidine-2-one (HA-966) which inhibits NMDA receptor function (Keith et al . , 1989), alters gamma-amino-butyric acid (GABA) and

tyrosine-related neurotransmitter activity (Reynolds et al . , 1989; Regunathan et al . , 1989) and the

hypothalamic-pituitary-adrenal axis (Morrow et al . , 1993) . Alterations in the hypothalamic-pituitary-adrenal axis (Demitrack et al . , 1991) and reduced basal plasma levels of the tyrosine derived metabolite,

3-methoxy-4-hydroxyphenylglycol (MHPG), have been found in CFS patients (Demitrack et al . , 1992). CFSUM1 may be related to these changes as it correlates with changes in tyrosine and phenylacetic acid suggesting an associated change in catecholamines. β-alanine is known to

competitively bind the GABA and glycine sites on neurone receptors (Wu et al . , 1993) and together with CFSUM1, these compounds may act as modulators of neurone

metabolism or receptor function and possibly alter the excitatory/inhibitory neurotransmission. These

interactions may be instrumental in the development of CFS whereby the concentration of these metabolites determines the severity of certain CFS symptoms. This is supported by the observation that the alteration in muscle function in CFS patients appears to be of central origin (Kent Braun et al . , 1993). CFSUM1 correlates with succinic acid and aconitic acid which suggest a perturbation of energy (TCA cycle) metabolism. The strong negative correlations of CFSUM1 with glutamic acid and alanine also suggest an adverse effect of CFSUM1 on nitrogen metabolism and inter-organ nitrogen transport. Methyl-proline analogues can

competitively inhibit proline oxidase (Trisch et al . , 1993) which is required for the conversion of proline to glutamate (Jones, 1985). N-methyl-pyrrolidone also has a structural homology with CFSUM1. It is an important solvent used in the pesticide industry for the

application of certain pesticides, for example Helix™. In the pharmaceutical industry it is also used to aid skin uptake of certain therapeutic products (Snyder, 1990) . It is interesting to note that

N-methyl-pyrrolidone induces alteration of nitric oxide production (Sax, 1984), inhibition of repolarization of retinal photo-receptors (Carricaburi et al . , 1980) and disturbance of cardiac rhythm and blood pressure (Baggio et al . , 1976). These effects suggest an association between modified pyrrolidine-ring metabolites and

CFS-like symptoms.

The structural association of CFSUM1 with known neuro- or excito-toxins, the relationship between CFSUM1 and symptom incidence as well as severity, and the correlation of CFSUM1 with other metabolic perturbations, makes it a very important metabolite in the diagnosis of a chronic disease state. Data correlating a variety of symptoms of chronic disease states and bacterial products and by-products is given in Examples 2 and 3, while

Example 7 correlates psychological symptoms in

particular. Although certain markers correlate strongly with certain aspects of a chronic disease state, i.e., the association between β-alanine and muscle pain

conditions, the present invention also includes the measurement of several markers simultaneously. As shown in FIG. 2, it is the interrelationship of one or more chronic disease conditions which leads to progressively more severe forms of chronic disease. The relative measurements of several markers can be used to determine the severity of the condition. In addition, as

demonstrated in Example 6, certain of the markers identified by the present invention are associated with particular species of staphylococci. Therefore, the measurement of these markers may be used to identify the infective agent, or a multiplicity of infection in a chronic disease state. If more than one microbe is identified as contributing to the chronic disease state, the treatment protocol may be adjusted accordingly. This may include a variety of antibiotics, or a combination of antibiotics with nutritional therapy or vaccines.

A strong association has also been made between chronic muscle pain and the carriage of human and horse membrane-damaging toxin producing staphylococci,

staphylococcal production of human and horse hemolytic toxins, increased incidences of infective events, and multi-organ signs and symptoms supportive of an infective aetiology. No significant association existed between chronic muscle pain and staphylococcal rabbit or sheep hemolyzing toxins, and staphylococcal enterotoxins A, B, C, D or toxic shock syndrome toxin-1. The data provided in Example 4 supports a strong association between staphylococcal human and horse hemolyzing toxins and chronic muscle pain conditions.

The association between toxicogenic staphylococcal carriage and chronic muscle pain may be the result of; 1) specific toxic effects, 2) toxin interactions with; a) other pathogens, b) environmental factors, c) host genetics, d) metabolic diseases, and/or 3) simply the induction of exoprotein production by staphylococci as a result of some host disease-related process. Biochemical changes associated with the carriage of staphylococcus species is presented in throughout the examples and, in particular. Example 6. The staphylococcal horse erythrocyte hemolysin has been identified in S.aureus but attempts to purify it were unsuccessful. In certain embodiments the preferred human hemolyzing toxins of the present invention are; 1) inhibited by lecithin and serum, 2) not effected by heating to either 60ºC or 100°C, and 3) cytotoxic in tissue culture indicating that they are either; 1)

S.aureus δ toxin, 2) S.epidermidis δ-like (Mollby, 1995) or ∈ (Ali & Haque, 1972) toxin or 3) similar

staphylococcal δ-like toxins. S.aureus δ toxin is a 26 amino acid peptide with an alpha helix structure, whilst S.epidermidis d-like or e toxin is a similar 25 amino acid peptide which has several amino acid residue

alterations (McKevitt et al . , 1950). These are similar immunologically but have been reported in one study not to be completely cross-reactive (Elek & Levy, 1950) . The staphylococcal species heterogeneity of these group of polypeptides is unknown but they are produced with great variability by many staphylococcal subspecies (Gemmell & Thelestam, 1981). This group of small polypeptides are high lipid soluble (Eriksson et al . , 1989) and are capable of; 1) forming cation selective voltage-dependent and -independent ion channels (Mellor et al . , 1988), 2) causing cell membrane lipid disturbances (Rydall &

MacDonald, 1992), 3) causing cell permeabilization

(Mollby, 1995), 4) activation of phospholipase A2 and the production of prostaglandins and platelet activating factor (PAF) (Rydall & MacDonald, 1992; Kasimir et al . , 1990), 5) causing disturbance of membrane receptor function (Umezawa et al . , 1980), and 6) acting as an enterotoxin. The high lipid solubility of δ (-like) toxins may facilitate their rapid absorption across intact skin or mucosal membranes and hence may initiate a low grade toxaemia without infection. This possibility may explain the difficulty in finding an onset-related etiological agent as this phenomena would not initiate any significant subject identifiable effect apart from the possibility of inflammation on a mucosal surface (e.g., sore throat, sinusitis, gut disturbances).

The present invention also relates to the

correlation of the onset of symptoms associated with a chronic disease state and an infective event by a microbe, for example, bacteria of the following species: Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. , Pseudomonas spp. , Bacillus spp. , Listeria spp. ,

Enterobacteriacaceae spp. , Spirochetes spp. , S. warneri , S. haemolyticus, S. xylosis, S. hominis, S. epidermidis or S. lugdunensis . Data for these correlations is presented in Examples 4 and 5.

C Assay Formats

The present invention also relates to assays of bodily fluids for the presence of microbes associated with chronic disease states. Assays include direct culturing of bodily fluids, either in liquid culture or on a solid support such as nutrient agar. A typical assay involves collecting a sample of bodily fluid from a patient displaying symptoms of a chronic disease state and placing the sample in conditions optimum for growth of the microbe. The determination can the be made as to whether the microbe exists in the sample in aberrant amounts. Further analysis can be carried out to

determine the hemolyzing properties of the microbe.

Biophysical methods such as, for example, high performance liquid chromatography (HPLC), fast protein electrophoresis (FPLC), capillary electrophoresis, electrospray mass spectrometry, affinity chromatography, may also be utilized to separate components of the sample of bodily fluid and generate a profile for analysis. The separation can be based on a selected bio-physical properties of the components such as size, charge, polarity, solubility or affinity. If the components to be analyzed are large biomolecules such as proteins or nucleic acids, electrophoretic techniques can also be used. In preferred embodiments the sample fluid is urine and the method of separation is GC-MS. An exemplary separation profile is shown in FIG. 1. One of skill in the art would also recognize that assays based on the derivatization of particular

substituents of the microbial organism or products, or by-products thereof, may also be utilized in the context of the present invention. Assays, for example, to derivatize primary amines such as reaction with opthaladehyde (OPA) to yield a blue colored fluorescent product may provide a simple detection method. Similar methods may be used to detect a single organism, product, or by-product, or may be expanded to include a variety of different reactions to provide unique derivatives for each of the organisms or products and by-products of the present invention. Relative measurements could then be made using, for example, colorimetric,

spectrophotometric, of fluorometric assays, dependent upon the nature of the derivatization agents employed.

The present invention relates to immunoassays for bacterial products or by-products. Antibodies and other toxin binding proteins (i.e., cell surface receptors) that recognize a product or by-product of a microbial organism selected from the group consisting of

Staphylococcus spp. , Candida spp. , Streptococcus spp. , Corynebacterium spp. , Bacteroides spp. , Fusiform spp. , Mycobacterium spp. , Clostridium spp. , Proteus spp. ,

Pseudomonas spp. , Bacillus spp. , Listeria spp. ,

Enterobacteriaceae spp. , Spirochetes spp. and the cell wall deficient variants thereof, are contemplated as targets in the immunoassays.

Immunoassays encompassed by the present invention include, but are not limited to those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (western blot).

Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such

techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the anti-microbial product-or by-product-specific antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA".

Detection may also be achieved by the addition of a second antibody specific for the desired antigen,

followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the desired antigen are immobilized onto the well surface and then contacted with the antibodies of the invention. After binding and appropriate washing, the bound immune complexes are detected. Where the initial antigen specific antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label.

Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labelled species before or during

incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described as below.

Antigen or antibodies may also be linked to a solid support, such as in the form of beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then

"coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface. In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of the antigen or antibody to the well, coating with a non-reactive

material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the clinical or biological sample to be tested under conditions effective to allow immune complex

(antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.

"Under conditions effective to allow immune complex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of

nonspecific background. The suitable conditions also mean that the

incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at

temperatures preferably on the order of 25° to 27ºC, or may be overnight at about 4ºC or so.

Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. Washing often includes washing with a solution of PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.

To provide a detecting means, the second or third antibody will have an associated label to allow

detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immune complex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex

formation, e.g., incubation for 2 hours at room

temperature in a PBS-containing solution such as

PBS-Tween.

After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H₂O₂, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e .g. , using a visible spectra spectrophotometer.

Alternatively, the label may be a chemiluminescent one. The use of such labels is described in U.S. Patent Nos. 5,310,687, 5,238,808 and 5,221,605.

A typical application of immunoprecipitation

involves the use of Staphylococcus aureus bacteria that have protein A expressed on the peptidoglycan cell wall. In this method, an antibody that recognizes the antigen of interest is quickly precipitated by the binding of the antibody to the protein A on the cells which are then easily precipitated by centrifugation (Kessler, 1981) . Alternatively, protein A-conjugated beads can be used in place of the S.aureus bacteria.

Assays for bacterial toxins, products or by-products of the present invention also can determined

normal/abnormal tissue distribution for diagnostic purposes. Methods for in vitro and in situ analysis are well known and involve assessing binding of

antigen-specific antibodies to tissues, cells or cell extracts. These are conventional techniques well within the grasp of those skilled in the art. For example, the antibodies of the present invention may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC) . Each tissue block may consist of 50 mg of residual "pulverized" prostate tumor. The method of preparing tissue blocks from these

particulate specimens has been successfully used in previous IHC studies of various prognostic factors, e . g. , in breast cancer, and is well known to those of skill in the art. (Abbondanzo et al . , 1990; Allred et al . , 1990; Brown et al . , 1990) Briefly, frozen-sections may be prepared by

rehydrating 50 ng of frozen pulverized prostate tumor at room temperature in PBS in small plastic capsules;

pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and pelleting again by centrifugation;

snap-freezing in -70°C isopentane; cutting the plastic capsule and removing the frozen cylinder of tissue;

securing the tissue cylinder on a cryostat microtome chuck; and cutting 25-50 serial sections containing an average of about 500 remarkably intact tumor cells.

Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting;

resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and embedding the block in paraffin; and cutting up to 50 serial permanent sections.

D. Antibodies

MAbs may be readily prepared through use of

well-known techniques, such as those exemplified in U.S. Patent 4,196,265. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g. , purified or partially purified

protein, synthetic protein or fragments thereof. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions. Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 × 10⁷ to 2 × 10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures

preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells, called "hybridomas."

Any one of a number of myeloma cells may be used and these are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions. One preferred murine myeloma cell line is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/₀ non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler & Milstein (1975;

1976), and those using polyethylene glycol (PEG) , such as 37% (v/v) PEG, by Gefter et al . (1977). The use of electrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies, about 1 × 10"⁶ to 1 × 10^-8. This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells

(particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium. The selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where

aminopterin or methotrexate is used, the media is

supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine. The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are

defective in key enzymes of the salvage pathway, e .g. , hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and

generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity

chromatography.

Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art. Monoclonal antibodies according to the present invention also may be monoclonal

heteroconjugates, i.e., hybrids of two or more antibody molecules. In another embodiment, monoclonal antibodies according to the invention are chimeric monoclonal antibodies. In one approach, the chimeric monoclonal antibody is engineered by cloning recombinant DNA

containing the promoter, leader, and variable-region sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene. The antibody encoded by such a recombinant gene is a

mouse-human chimera. Its antibody specificity is

determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.

In another embodiment, monoclonal antibodies

according to the present invention is a "humanized" monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the

framework regions of their murine counterparts.

"Humanized" monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods.

As stated above, the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art. Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e .g. , feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for large scale hybridoma cultivation under tissue culture

conditions are known in the art and include homogenous suspension culture, e . g. , in an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture.

Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo . Cell clones are injected into mammals which are histocompatible with the parent cells, e .g. , syngeneic mice, to cause growth of

antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as

Pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide

synthesizer, or they may be produced manually using techniques well known in the art. The monoclonal conjugates of the present invention are prepared by methods known in the art, e . g. , by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.

Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as ³H, ¹²⁵I,

¹³¹I ³²p , ³⁵S , ¹⁴C , ⁵¹Cr, ³⁶Cl , ⁵⁷Co , ⁵⁸Co , ⁵⁹Fe , ⁷⁵Se, ¹⁵² Eu and ⁹⁹mTc, are other useful labels which can be conjugated to antibodies. Radioactively labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium-"m by ligand exchange process, for example, by reducing

pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e .g. , by incubating pertechnate, a reducing agent such as SNC1₂, a buffer solution such as sodium-potassium

phthalate solution, and the antibody.

E. Purification of Markers

Purified microbial products and by-products also are encompassed by the present invention. For purposes of the present invention, a product is defined as purified, where at least about fifty percent of a given sample by weight is comprised of the product. The lipid soluble exoproteins, bacterial products and by-products of the present invention may be isolated by a variety of isolation techniques well known in the art. MacDonald et al . (1989). Examples include, but are not limited to gas chromatography, ion exchange chromatography, gel filtration, size exclusion chromatography, HPLC, FPLC, density gradient centrifugation and gel electrophoresis. In situations where the purified protein is to be used for the production of antibodies, it may be desirable to further purify the antigen using dialysis or desalting columns for example.

F. Psychological Testing

The present invention also contemplates the

connection between psychological symptoms commonly associated with chronic disease states and the

measurements of bacterial products and by-products.

Psychological status was evaluated using the Hopkins Symptom Checklist-90-Revised(SCL-90-R) as discussed in Examples 1 and 2. An index of cognitive impairment is constructed by evaluation of responses to questions from within the SCL-90-R. Some Example questions ask the patient if they have trouble performing the following functions: trouble remembering things, having to do things very slowly to ensure correctness, difficulty making decisions, your mind going blank or trouble concentrating (Byrne et al . , 1985). In certain

embodiments, a visual analogue pain scale (VAPS) of average pain intensity is also evaluated.

Example 5 demonstrates a significant association between carriage of toxicogenic staphylococci which produce human and horse hemolyzing toxins and 29 muscle pain-related questions in the SCL-90-R. This correlation indicates that a negative response to 9 SCL-90-R somatic questions could be used to select a clinically and psychologically asymptomatic control group who were not carriers of toxicogenic staphylococci.

III. Therapies for Chronic Disease States

Because certain microbial agents that contribute to chronic disease states have been identified, the present invention provides, for the first time, a meaningful approach to the treatment of these chronic disease states. Fortunately, because there are numerous

therapeutic regimens that are routinely applied to the treatment of microbial infestations, it is expected that these regimens can be applied successfully to the treatment of microbially-induced chronic disease states. The following are examples of anti-microbial therapies according to the present invention.

In one embodiment, the treatment will be directed against the microbe itself in an attempt to reduce or eliminate its colonization of the host. In another embodiment, it will be desired to block the pathologic effects of the microbe by eliminating or neutralizing products or by-products of the microbial colonization. In yet another embodiment, the treatment will be

ameliorative in the sense that the general state of health of the host will be augmented, in a particular fashion, without focusing on the microbe, its products or by-products. A. Vaccines and Active Immunity

One of the strongest defenses against microbial infestation is the hosts own immune system. In some instances, it may prove effective to "prime" the immune system so that the immune response against a certain microbe is more rapid, more sensitive or more intense than it would otherwise be. By providing a potential host with a vaccine that stimulates the immune system in this manner, active immunity is generated. Active immunity is defined, for the purpose of this application, as the generation of an immune response in an animal for the purpose of protecting that animal from a microbe that is associated with chronic disease states.

In one form, it will be desired to provide a vaccine comprising killed microbes. The preparations may be "killed whole cell" preparations, meaning that they are non-living, unfractionated samples from a microbe

culture. Alternatively, the preparation may be a

"disrupted cell membrane" preparation which is partially purified in the sense that intracellular structure such as nucleic acids and mitochondria have been eliminated. These vaccines may either be monospecific (a single organism) or polyspecific (multiple organisms).

A more specific form of immunization involves the use of particular, purified microbial products or

by-products. The description of purification methodolocfy is set out in greater detail above. Following

purification of a bacterial product or by-product to an acceptable degree (which is generally in excess of 50% of the total amount of the sample), the product or

by-product can be administered to the animal in the form of a vaccine.

The antigen composition is prepared by mixing, preferably homogeneously mixing, at least one microbe preparation or antigen with at least one pharmaceutically or veterinarally acceptable carrier, diluent, or

excipient using standard methods of pharmaceutical or veterinary preparation. In preferred embodiments of the invention, the animals to be immunized are mammals such as cats, dogs, horses and, more particularly, humans, although there is no limitation other than that the subject be capable of mounting an immune response of some kind.

The immunogenicity of microbial products or

by-products may vary and, therefore, it may be desirable to couple the immunogen to a carrier molecule. Exemplary carriers are keyhole limpet hemocyanin (KLH) and human serum albumin. It also may be desirable to include in the vaccine any of a number of different substances referred to as adjuvants, which are known to stimulate the appropriate portion of the immune system of the vaccinated animal. Suitable adjuvants for the

vaccination of animals include, but are not limited to oil emulsions such as Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol

52:Montanide 888 (Marcol is a Trademark of Esso,

Montanide is a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), mineral gels such as aluminum hydroxide, aluminum phosphate, calcium phosphate and alum, surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N,N'-bis(2-hydroxyethyl)-propanediamine,

methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose

dimycolate.

The microbe preparations or bacterial products or by-products of the present invention also can be

administered following incorporation into liposomes or other micro-carriers, or after conjugation to

polysaccharides, proteins or polymers or in combination with Quil-A to form "iscoms" (immunostimulating

complexes). These complexes can serve to reduce the toxicity of the antigen, delay its clearance from the host and improve the immune response by acting as an adjuvant. Other adjuvants suitable for use in the present invention include INF, IL-2, IL-4, IL-8 and other immunostimulatory compounds. Further, conjugates

comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT (see PCT/AU87/00107) may prove advantageous. Routes of administration, dosages to be

administered, and frequency of injections are all factors which can be optimized using ordinary skill in the art. The antigen composition may be administered

intravenously, subcutaneously, intranasally, orally, intramuscularly, urethrally, vaginally, rectally,

topically or via any other desired route. Of particular interest will be the topical, oral, urethral and

gastrointestinal routes as many of the organism of interest here colonize the surfaces of the skin, gut and urinary tract.

Typically, the initial vaccination is followed some weeks later by one or more "booster" vaccinations, the net effect of which is the production of vigorous

cellular and humoral immune response. The need for, or the appropriate timing of, subsequent boosters can be evaluated by measuring the immune response of the subject following each immunization. Animals would generally require two vaccinations 4-18 weeks apart and additional vaccinations annually or more frequently as desired.

One focal point in the area of vaccines will involve the immunization of toxins, particularly peptide toxins of Staphylococcal origin. One of the difficulties of immunizing an animal with a toxin is the toxic effect that the toxin will exhibit in vivo. In order to

overcome this problem, it may be necessary to reduce or eliminate the toxicity of the toxin. This may be

accomplished in several different ways. First, it may be desirable to denature the peptide so that higher order (secondary, tertiary, etc.) structure of the peptide is lost. This can be accomplished by heating or treating the peptide with a denaturing agent such as an acid or a base. A particular form of chemical denaturation

involves the use of thiol-containing reducing agents that disrupt disulfide bonds in the peptide. A variation on this approach is the actual fragmentation of the peptide by chemicals or peptide-cleaving enzymes, known as endoproteases.

A second way of reducing or eliminating toxicity is by conjugating another molecule to the toxin or,

alternatively, conjugating the toxin to another molecule. This may block particular "toxic" sites on the toxin or reduce the toxins accessibility to molecules with which the toxin interacts via steric hinderance. It also may block uptake and/or internalization of the toxin by host cells, or transport of the toxin within the cell once internalized.

Yet another way in which peptide toxins can be modified is by site-directed mutagenesis. Peptides are made up of repeating units of amino acids. By altering the DNA encoding the peptide such that one or more of these amino acids are substituted by others, it is possible to introduce relatively subtle changes into the primary and, hence, secondary and tertiary structure of the toxin. This also can be accomplished by generating the peptides synthetically if their length is less than about 75 amino acids. In this way, it may be possible to eliminate the toxicity of the molecule without destroying so much of its structure that it's antigenic character is lost. Such substitution mutants would be able to elicit a protective immune response without causing a toxic reaction.

In creating such substitution mutants, there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent

biological activity. Substitution mutants are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.

Particular embodiments encompass variants that have one, two, three, four, five or more variations in the peptides sequence. Of course, a plurality of distinct

proteins/peptides with different substitutions may easily be made and used in accordance with the invention.

An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.

The hydropathic index of amino acids also may be considered. Each amino acid has been assigned a

hydropathic index on the basis of their hydrophobicity and charge characteristics, these being: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);

alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline

(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic amino acid index in structural integrity of a protein is generally

understood in the art (Kyte & Doolittle, 1982) . it is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±l are particularly preferred, and those within ±0.5 are even more particularly preferred.

It also is understood in the art that the

substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the substitution peptide thereby created is intended for use in immunological embodiments, as in the present case.

U.S. Patent No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its imrounogenicity and antigenicity, i .e . , with a biological property of the protein.

As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);

aspartate (+3.0 ± 1); glutamate (+3.0 ± l) ; serine

(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5);

histidine (-0.5); cysteine (-1.0); methionine (-1.3);

valine (-1.5); leucine (-1.8); isoleucine (-1.8);

tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose

hydrophilicity values are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

Having described substitution mutants of toxic peptides that are suitable for use as immunogens, it also is contemplated that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the toxin peptides of the invention. The generation of a structural equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such

sterically similar constructs fall within the scope of the present invention. B. Passive Immunity

Passive immunity is defined, for the purposes of this application, as the transfer to an organism of an immune response effector that was generated in another organism. The classic example of establishing passive immunity is to transfer antibodies produced in one organism into a second, immunologically compatible animal. By "immunologically compatible," it is meant that the antibody can perform at least some of its immune functions in the new host animal. More recently, as a better understanding of cellular immune functions has evolved, it has become possible to accomplish passive immunity by transferring other effectors, such as certain kinds of lymphocytes, including cytotoxic and helper T cells, NK cells and other immune effector cells. The present invention contemplates both of these approaches. Antibodies, antisera and immune effector cells are raised using standard vaccination regimes in appropriate animals, as discussed above. The primary animal is vaccinated with at least a microbe preparation or one bacterial product or by-product according to the present invention, with or without an adjuvant, to generate an immune response. The immune response may be monitored, for example, by measurement of the levels of antibodies produced, using standard ELISA methods.

Once an adequate immune response has been generated, immune effector cells can be collected on a regular basis, usually from blood draws. The antibody fraction can be purified from the blood by standard means, e . g. , by protein A or protein G chromatography. In an

alternative preferred embodiment, monoclonal

antibody-producing hybridomas are prepared by standard means (Coligan et al . , 1991). Monoclonal antibodies are then prepared from the hybridoma cells by standard means. If the primary host's monoclonal antibodies are not compatible with the animal to be treated, it is possible that genetic engineering of the cells can be employed to modify the antibody to be tolerated by the animal to be treated. In the human context, murine antibodies, for example, may be "humanized" in this fashion.

Antibodies, antisera or immune effector cells, prepared as set forth above, are injected into hosts to provide passive immunity against microbial infestation. For example, an antibody composition is prepared by mixing, preferably homogeneously mixing, at least one antibody with at least one pharmaceutically or

veterinarally acceptable carrier, diluent, or excipient using standard methods of pharmaceutical or veterinary preparation. The amount of antibody required to produce a single dosage form will vary depending upon the

microbial species being vaccinated against, the individual to be treated and the particular mode of administration. The specific dose level for any

particular individual will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the individual, time of administration, route of administration, rate of excretion, drug

combination and the severity of the microbial

infestation. The antibody composition may be administered intravenously, subcutaneously, intranasally, orally, intramuscularly, vaginally, rectally, topically or via any other desired route. Repeated dosings may be necessary and will vary, for example, depending on the clinical setting, the particular microbe, the condition of the patient and the use of other therapies.

C. Antibiotic Therapy Because of the wide variety of antimicrobial agents available to the clinician, including a host of

antibiotics and antivirals, there are many different chronic disease states that can be treated using

relatively standard protocols, i.e., using proven drugs with known routes, dosages and treatment regimen. On the other hand, there is some evidence that ill-advised use of antibiotics may, in fact, contribute to the onset or aggravation of chronic disease states, presumably because the natural microbial flora is upset by certain anti-bacterial treatments.

Typically, antibiotic treatments are performed by first selecting an antibiotic and then monitoring the effect on the microbe, primarily to assess the

sensitivity to the antibiotic. This sort of approach may, in fact, contribute to chronic disease. The present invention proposes, instead, a specific regimen that seeks to maximize microbiocidal action without the development of resistance. Another problem that is to be avoided is the selective mistargeting of cell wall- containing bacteria. Because chronic disease states are, at least in part, caused by cell wall-deficient bacteria, the use of cell wall anti-microbials is inappropriate and may exacerbate the pathologic situation.

An antibiotic therapy according to the present invention will employ at least two different antibiotics - one that targets microbial ribosomes and one that targets microbial DNA. These agents may be used

simultaneously or alternatively. The therapy preferably is a long term one (3 months, 6 months, 9 months, one year, or more) and, therefore, the antibiotics should be rotated on a weekly basis. For example this may be accomplished by using drug A in week one, drug B in week two, drug A in week three, drug B in week four, and so on. Alternatively, drugs A and B can be used in week one, drugs C and D can be used in week two, drugs A and B can be used in week three, drugs C and D can be used in week four, and so on. Various other formats also may be followed. Routes of administration, doses, dosage formulations and other clinical parameters may be established by the clinician based on standard criteria including the age and general health of the patient, the type of chronic disease state, the duration of treatment, toxicity of the drug(s), and other relevant factors. Agents that act on bacterial ribosomes include macrolides, such as

erythromycin, aminoglycosides, such as gentamycin, and tetracycline-related compounds including long-lasting forms such as vibramycin and minocycline. Agents that act on bacterial DNA include quinolones such as

norfloxacin and ciproflaxin. D. Nutritional Therapy

As a result of the microbial infections that cause chronic disease states, various toxins and metabolites of the microbes interfere with receptor function and

enzymatic pathways in the host organism's cells.

Metabolic pathways of the organism are adversely

affected. However, the effects of the toxins and/or metabolites may be overcome by providing nutritional supplements that either (i) replace host cell metabolites that are lacking because of impaired metabolic function or (ii) overcome the effects of the bacterial toxins or metabolites by "out-competing" these molecules. The present invention contemplates the use of a variety of nutritional supplements. For example,

vitamins, amino acids, and fatty acids are desirable nutritional compounds that may be employed. Amino acids preferred in this regard are glutamine, glycine, leucine, isoleucine, proline, and phenylalanine. Fatty acids include essential fatty acids, such as those in evening primrose oil and fish oil. Also contemplated are

vitamins and cations Mg²⁺ and K⁺. E. Combinations

As a practical matter, it may be advisable to use combinations of treatment regimens, to achieve optimal therapeutic effects. For example, it may be desired to use a vaccine or passive immunization in combination with antibiotics or nutritional therapy, or both. It may be advisable to use nutritional therapy with antibiotics or with vaccines (or both). It also may be desired to use antibiotic with nutritional therapy or with vaccines (or both) . IV. Kits

According to another aspect of the present

invention, different embodiments of the invention may be provided in the form of a kit. The kit comprises one or more receptacles for reagents described above. The kit may further comprise reagent solutions, such as antigen composition, antibody compositions, enzyme and enzyme substrate solutions, and other solutions useful in performing analytical tests, such as buffer solutions, growth media and washing solutions.

Preferably, the solutions are provided in separate, sealed containers in premeasured amounts useful for performing a single assay. The kits also may provide supports upon which certain reactions can be performed including culture dishes, 24-well and 96-well culture trays. Also, supports suitable for dipping into chambers such as test tubes are contemplated. The kit also may include caps for the containers so that incubation and mixing is possible. The shape and size of the support and the containers may be adapted for a variety of purposes. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the

techniques disclosed in the examples which follow

represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1

V. METHODS

A. Study Subjects

As part of a clinical study on the aetiology of chronic orofacial pain, sequential pain subjects referred for assessment were examined. Twenty study patients having a chronic disease state (patients) and forty-five control subjects (subjects), having no indication of a chronic disease state, were assessed over a six month period. The patient group comprised patients that had previously been diagnosed as having a chronic disease state and met the chronic disease state research

criteria. Patients and subjects were given a

questionnaire (CPRU), which was specially developed for the inventor's studies of chronic pain/fatigue illnesses, and a System Check List-90-Revised (SCL-90-R)

psychological inventory. The questionnaire including pain distribution diagrams, and questions involving medical history, signs and symptoms, duration and

severity of condition. All questionnaires were checked by one investigator and the patients questioned about any aspects of the questionnaire that they had trouble completing.

B. Patient Microbiological Sample Collection

After detailed instruction, subjects collected nasal and in the female study participants a low vaginal swab. These swabs were transported in Stuart medium. C. staphylococcal Enterotoxin and TSST-1 Assay

The nasal and genital samples were inoculated onto horse blood agar and incubated at 37ºC in a humidified incubator supplemented with 10% CO2. Staphylococcal isolates were selected by colony morphology and coagulase was tested by the tube method. Staphylococcal isolates were grown in 5mL brain heart infusion broth and

incubated at 37°C in a humidified incubator supplemented with 10% CO2 for 24 hours. The cultures were centrifuged at 2,5009 at 4°C for 20 minutes. Staphylococcal

enterotoxins A, B, C, D (SEA, SEB, SEC, SED) and toxic shock syndrome toxin-1 (TSST-1) were detected using the reversed passive latex agglutination toxin test kits (SET-RPLA and TST-RPLA respectively, Oxoid, Basingstoke) . An equal volume (4uL) of staphylococcal supernatant and either latex particles sensitized to TSST-1, SEA, SEB, SEC or SED, or a paired negative control, were mixed in a Kova slide compartment and refrigerated at 4°C for 18-24 hours. These were observed both visually and by phase contrast microscopy.

D. Staphylococcal Haemolysin Assay Membrane-damaging toxins were assessed by haemolysis assays with the use of rabbit, sheep, horse and human type O erythrocytes. Supernatants from overnight growth of staphylococcal isolates were added to triple washed erythrocytes, suspended in 1mm magnesium phosphate buffered saline (0.5 KH2PO4 and 0.5M K2HPO4) to give a final concentration of 1% (v/v). An equal volume of staphylococcal supernatant was added to each of the four erythrocyte pellets and incubated at 35°C for 30 minutes. The sheep erythrocyte suspensions were further

refrigerated at 4°C for 24 hours to allow for the

hot/cold haemolysis effect. Samples were centrifuged at 3,500 for 5 minutes and the absorbance (54lira) was measured. The results were expressed as the percentage of the absorbance of a control tube containing totally lysed erythrocytes. The different haemolysis assays allowed detection of staphylococcal a-like (rabbit), p-like (sheep), y-like (rabbit, sheep and human), or a-like toxin (human) and horse haemolytic toxin. The haemolysis rate per study participant was determined by the staphylococcal isolate with the highest haemolysis rate.

E. Definition of Haemolysin Toxicity

An asymptomatic group was used to define the levels of haemolysis that determine symptom expression. Any non-CFS subject who scored zero in the SCL-90-R questions fourteen (fatigue) and forty-two (muscle soreness) or less than two positive responses to somatization

dimension (questions 1, 4, 12, 27, 40, 48, 49, 52, 53, 56, 58) not including question 42, was classified as asymptomatic. These subjects, by definition, do not have fatigue or muscle pain or combinations of headaches (Q1), muscle or lower back pain and weakness (Q27, Q56, Q58), paraesthesia (Q52), dizziness (Q4), febrile-like events (Q49), breathlessness (Q48) and nausea (Q40) . These symptoms are representative of the core symptoms of CFS definitions. Importantly the SCL-90-R is based on the presence or absence of symptoms within the preceding 7 days and has a scalar score, and is thus gives an

appropriate time and severity-based response in

relationship to the types of staphylococcal toxins present in the sites sampled.

F. GCMS technique A 10 mL aliquot of each sample was centrifuged at 1,500xg for five minutes at 4°C and a 200pL aliquot was transferred to a derivatization tube and freeze dried for eighteen hours. The freeze dried supernatant material was then reacted to form the

N(O,S)-heptafluorobutyryl-isobutyl (HFB-isobutyl) derivatives for analysis by GC-MS.

Derivatized samples were separated using a Hewlett Packard 5890 Series II gas chromatograph (GC) and

detected by a Hewlett Packard 5971A mass selective detector (MSD) . The data were stored and processed on a Hewlett Packard Unix-based Chemstation. The GC was fitted with a 25.0m, 0.20 mm internal diameter HP1 fused silica capillary column (film 0.33pm) and split/splitless injector. The GC-MS was run with the following settings: injector temperature 300ºC, temperature program 80-300°c at 3°C min-1, with a 2 minute hold at 80ºC and a 10 minute hold at 300°C. The mass spectrometer was set to scan from 45-650 atomic mass units every second. An injection volume of 0.5 microlitres was used. G. Urine Specimens and GC-MS Identification

The first morning urine specimens were collected by the patients and subjects on the day of the second visit. The urine was refrigerated and processed within

twenty-four hours of collection and 10 mL aliquots were processed as described.

Twenty-eight urine peaks were selected for

examination after confirming a linear detector response by the mass spectrometer. Where possible, the peaks were identified by HP-UX Chemstation computer search of user-generated reference libraries (incorporating

retention indices and mass spectra) and the WILEY

Database^™. The peaks were numbered one to twenty-eight in order of retention time and those peaks that could not be identified were allocated a reference code (either UM or CFSUM) . The percentage abundances of the twenty-eight peaks were summarized to assess qualitative changes in urinary composition.

H. staphylococcal Isolate Supernatants and GCMS Identification

Ten mL aliquots of the supernatants from overnight growth of staphylococcal isolates in X broth were

derivatized as described. Where possible, the peaks were identified by HP-UX Chemstation computer search of user-generated reference libraries (incorporating

retention indices and mass spectra) and the WILEY

Database™. The peaks were compared with those found in the urine of the study participants using retention time and mass spectra.

I. Group Comparisons

The twenty chronic disease state patients were compared to: 1) the total control subjects; 2) to a group of control subjects who were CFSUM1 positive (carrier group); and 3) a group of control subjects who had no detectable CFSUM1 (non-carrier group). The effect of each staphylococcal toxin was assessed by comparison of the total study subjects (n=65) by: 1) the presence or absence of the various enterotoxins and TSST-1; 2) division of subjects around the defined haemolysis levels determined for each toxin or toxin combination; and 3) division of subjects into groups determined by the presence and absence of the peaks.

J. statistical Analysis

The urine and haemolysis data (both arcsin

transformed) and clinical data were analyzed using t-tests, Chi-square, Fischer exact probability, Pearson product-moment and Spearman rank order correlations, anova, discriminant function and multiple regression analysis (significance p<0.05). The discriminant

variables were defined as positive or negative if they increased or decreased in incidence of relative abundance in relationship to the variable being measured.

Sensitivity (Sen), defined as the percentage incidence of true positives in a test group, and specificity (Spec) , defined as the percentage of true negatives in the comparison group, were determined. These data were processed using Access^™ (Ver. 1.1, Microsoft), Excel^™ (Ver. 4.0, Microsoft) and the computer statistical package Statistica^™ (Ver. 4.5, Statsoft, Tulsa) .

VI. RESULTS A. Subject Characteristics

Table 2 shows the age and sex characteristics of the participants in the chronic disease state patient group, the control subjects in the carrier (CFSUM1-positive) and non-carrier (CFSUM1-negative) groups. No significant sex or age differences were noted in any of the groups. All chronic disease state patients complied with the Oxford and American CDC CFS symptom criteria. B. Determination of the Membrane Damaging

Haemolysis Levels

Eight of the forty-five control subjects were found to be asymptomatic by non-responses to the SCL-90-R somatization dimension questions. These subjects also did not respond to any symptom questions in the pain questionnaire. The mean and standard error of

staphylococcal haemolysis for the membrane-damaging toxins in this defined asymptomatic group were: rabbit (0.72±1.6%); sheep (8.0±6.8%); human (0.57±0.5%) and horse (3.2±1.2%). The haemolysis rates for division of haemolytic toxin activity were thus decided to be; 1) rabbit 5%; 2) sheep 10%; 3) human 5%; and 4) horse 7%. Subjects were grouped for analysis of the importance of the haemolytic toxins by grouping subjects above and below the haemolysis rates as positive or negative carriers.

C. incidence of staphylococcal Species and Toxins

Table 3 shows the numbers of subjects and

staphylococcal isolates tested with the subjects divided into chronic disease state patients, control subjects and the CFSUM1-carrier and non-carrier subgroups of the control subjects. Table 3 also shows the incidence of the staphylococci species, enterotoxins, toxic shock syndrome toxin (TSST-1) and of the various haemolysins.

Staphylococcus spp. that produce: 1) a horse red blood cell haemolysin; and 2) a novel toxin, CFSUM1

(amino-hydroxy-N-methyl-pyrrolidine), have an increased incidence (16/20 - 80% v 14/45 - 31.1%; p<0.0005) in patients who comply with the chronic disease state definition. Study participants who carried these

staphylococci had increases in the relative abundance of CFSUM1 in their urine (p<0.000005) compared to

non-carrier subjects. Carriage of these staphylococci was associated with an increased incidence of the core diagn *ostic symptoms of CFS. Subjects positive for the two toxins had increases in urinary abundance of the tricarboxylic acid intermediates, aconitic and succinic acid, the amino acid tyrosine and reductions in the metabolite CFSUM2 and the amino acids glutamic acid, alanine, isoleucine and proline. The carrier subjects who comply with the chronic disease state definition had increases in CFSUM1 and β-alanine and reductions in

Lysine compared to the carriers who did not comply with the CFS definition. Tables 4 through 13 present further correlations between incidence of microbial agents, products and by-products and chronic disease states. VII. DISCUSSION

The data presented in this study indicate that

CFSUM1 (amino-hydroxy-N-methyl-pyrrolidine) is the product of Staphylococcus lugdunensis which also produces a horse red blood cell haemolysin. These data show that CFSUM1 has a strong relationship to the pathophysiology of a chronic fatigue illness which may effect up to 45% of the community which is the product of a specific group of staphylococci.

EXAMPLE 2

This example involves the correlation between alterations in metabolism and homeostasis, changes detected in the excretion of intermediary metabolites and waste products in the urine, which may indicated altered metabolic activities. Urine samples from patients with a chronic disease state and control subjects having no chronic disease state, were screened using capillary gas chromatograph - mass spectrometry (GC-MS) .

I. MATERIALS AND METHODS

A. Study Subjects

Twenty-two chronic disease state patients (patients) and 45 age and sex-matched control subjects were

recruited over a six month period. The chronic disease state group comprised patients that had previously been diagnosed as having a chronic disease state and met the Oxford chronic disease state research criteria (Sharpe et al . , 1991). All subjects were provided with a

questionnaire addressing onset events and symptom

incidence and a protocol for collection of a first morning urine sample at the first consultation. All subjects were asked to list the drugs and naturopathic remedies taken and the dietary changes that they had made within the preceding four weeks. The age and sex-matched control subjects were recruited from relatives of the patients and from unrelated subjects. None of the control subject reported fatigue that affected their lives. All samples were coded for analysis and data processing. On completion of all laboratory analyses the data were decoded and grouped for statistical analysis. Psychological status was assessed with the Hopkins

Symptom Checklist-90-Revised (SCL-90-R). The SCL-90-R is scored in 9 dimensions, including somatization,

depression and anxiety, with sex and age normalized scales with a score of >63 considered to be consistent with psychopathology (Derogatis, 1975).

B. Urine Specimens and GC-MS Identification

The first morning urine specimen was collected by each subject on the day of the second visit. The urine was refrigerated (not frozen) and processed within twenty-four hours of collection. A 10 Ml aliquot was centrifuged at 1,500g for five minutes at 4°C and a 200μL aliquot was transferred to a derivatization tube and freeze dried for eighteen hours.

The freeze dried urine material was then reacted to form the N(O,S)-heptafluorobutyryl-isobutyl (HFB-isobutyl) derivatives for analysis by GC-CMS (MacKenzie & Tenaschuk, 1979a; MacKenzie & Tenaschuk, 1979b). Two hundred microliters of HCL-isobutanol were added to the freeze dried sample and heated at 110°C for one hour. After cooling and evaporation by high purity nitrogen gas, the sample was freeze dried for thirty minutes.

Ethyl acetate (50μL) and heptafluorobutyric anhydride (20μL, HFBA) were added and the sample heated at 110°C for thirty minutes. After cooling, the sample was evaporated with nitrogen gas and freeze dried for five minutes and then redissolved in 200/LIL of ethyl acetate. Derivatized urinary metabolites were separated using a Hewlett Packard 5890 Series II gas chromatograph (GC) and detected by a Hewlett Packard 5971A mass selective detector (MSD) (Walsh et al . , 1995). Quality control was achieved by running a selected sample source every week with each batch of analyses to monitor extraction

efficiency and machine response, and quantitative

responses were assessed by an external standard strategy. Internal standard strategies were not used because these standards may co-elute with novel disease related metabolites in the chromatogram. Where possible, the peaks were identified by HP-UX Chemstation computer search of user-generated reference libraries

(incorporating retention indices and mass spectra) and the WILEY Database^™. The peaks were numbered one to twenty-eight in order of retention time and those peaks that could not be identified were allocated a code as indicated in the text. The reference spectra and retention indices of coded peaks were added to the use generated reference library for identification of peaks in subsequent analyses. Creatinine is routinely used as an internal

reference compound in urinary studies. Creatinine could not be assessed by this GC-MS method, but 3-methylhistidine, which was measured in this study, provides an acceptable alternative internal reference compound (Rudman, 1980) . However, as some studies report muscle, cytokine and immune anomalies in the patients (Hyde et al . , 1992), it was therefore necessary to determine whether the use of 3-methylhistidine as an internal reference compound is valid in chronic disease state studies. The philosophical basis of an internal reference requires that: 1) it has a constant excretion rate and is therefore not correlated with changes in other excretion products; 2) that it does not vary between the groups tested in the study; 3) that it does not vary in association with symptom expression; and 4) that it is not associated with changes in the instruments used to assess the patients. 3-methylhistidine was therefore assessed for correlation with urinary

metabolites, symptom incidence and the SCL-90-R

responses. Statistical analysis of the inventors' data revealed that 3-methylhistidine significantly correlated with levels of tyrosine, 1-methylhistidine and lysine (p<0.05) in the patients, whereas in control subjects, 3-methylhistidine correlated with glycine, aconitic acid, leucine, 1-methylhistidine and ethanolamine (p<0.05). This indicates that the excretion of 3-methylhistidine is not constant in relation to other metabolites and that the metabolic associations were different in patients compared to the control subjects. In addition, 3-methylhistidine was correlated with symptom expression including fever, lymphadenia, headaches and chest pain as well as the SCL-90-R obsessive compulsive dimension scores in patients exhibiting symptoms of a chronic disease state. In contrast, no such correlations were observed for 3-methylhistidine in the control subjects. It was concluded therefore that the use of urinary 3-methylhistidine (or creatinine) as a urinary reference constant is not valid in chronic disease state studies of this type and would introduce significant analysis errors. Consequently, the metabolite data was presented as the percentage abundance of the total peak area of all the peaks assessed in each urine sample.

C. Statistical Analysis The urine data (arcsin transformed) and clinical data were analyzed using t-tests, Pearson product-moment correlations, multivalent and one-way analysis of

variance (MANOVA and ANOVA) and forward stepwise

discriminant function or multiple regression analysis. Correction for statistical multiplicity occurred where applicable. Group differences were also assessed using Hotellings T². These data were processed using Access™ (Ver. 1.1, Microsoft), Excel™ (Ver. 4.0, Microsoft) and Statistica™ (Ver. 45., Statsoft, Tulsa). II . RESULTS

A. Subject Characteristics Twenty-two patients with a chronic disease state were recruited and two were excluded because one was taking medication within 7 days prior to specimen

collection, while the urine specimen of the other had bee inappropriately collected. No drug-related metabolites were detected in the urine samples. Table 14 shows the characteristics of the patients (n=20) and the comparison group of control subjects (n=45). No significant age or sex differences were noted between the groups. The inventors' study population was consistent with a level II outbreak as defined by Briggs & Levine (1994). All the patients complied with the Oxford CFS definition criteria (Sharpe et al . , 1991) and the American CDC case definition (Holmes et al . , 1988). B. Urinary Metabolite Changes

FIG. 1 shows the total ion chromatogram of a urine sample from a CFS patient. The corresponding peak numbers, identity, retention time, retention indices and mean percentage composition values for the twenty-eight peaks analyzed in this study are summarized in Table 15. Peaks that could not be identified were allocated a urinary metabolite (UM) code and those which were

significantly different between the CFS and non-CFS groups were allocated a chronic fatigue symptom urinary marker (CFSUM) code. Eight of the twenty-eight urinary compounds had significantly altered relative abundances when comparing the CFS and non-CFS groups (Table 15) . No differences were found between the groups for the

concentrations of the branched chain amino acids, isoleucine or leucine, and the protein catabolism indicator 3-methylhistidine.

Multivariant analysis of the urine data by the Hotellings T² test indicated that the patient group had a significantly different urinary excretion profile compared with the control subject group (p<0.004).

Forward stepwise discriminant function analysis also confirmed the difference in urine profiles between the two groups (Model: Wilks' lambda = 0.37012, F=6.6763, p<0.00001) and this analysis identified the most

important metabolites for differentiating between the groups. The most important discriminating metabolite was peak 12 (FIG. 1) which was identified as amino-hydroxy-N-methyl-pyrrolidine and will be referred to as CFSUM1 (p=0.0001). The mass spectrum of the HFB-isobutyl derivative of this metabolite is shown in FIG. 1 with the major ion at m/z=84 which is characteristic of N-methyl-pyrrolidine derivatives such as N-methyl-proline. The molecular ion at m/z-354 is equivalent to the molecular weight of the HFB-isobutyl derivative of amino-hydroxy-Nmethyl-pyrrolidine. The structure of amino-hydroxy-Nmethyl-pyrrolidine can be compared with similar

pyrrolidine derivatives such as N-methyl-pyrrolidone, proline and n╌methyl proline. The second most important discriminating metabolite was identified as β-alanine and the third was peak 2, which could not be identified by its mass spectrum and is referred to as CFSUM2. CFSUM1 was found in seventeen of twenty CFS patients compared with twenty-one of forty-five non-CFS subjects (85% v 46.7%; p<0.004). β-alanine did not have a

significantly different incidence (18/20 - 90% v 34/45 -75%). All patients and subjects had CFSUM2. No

significant sex or age differences were found for

incidence or relative abundance for CFSUM1, CFSUM2 or β- alanine. All the patients had either CFSUM1 or β-alanine whilst six of the 45 control subjects did not have either metabolite. Correlation statistics were used to assess the metabolite alterations in the entire study group in relation to the prime discriminant metabolites, CFSUM1, β-alanine and CFSUM2, which are summarized in Table 16. The correlation coefficients were also determined for each group to further assess differences in the metabolic interactions measured in the CFS patients compared with those measured in the control subjects. A number of metabolites were correlated with the level of CFSUM1 in the CFS patients, but exhibited no such relationship in the non-CFS subjects, including phenylacetic acid (CFS, r=+0.8962 p<0.001 v's non-CFS, r=+0.0045; p=0.9) and proline (CFS, r=-0.6115 p<0.001 v's non-CFS, r=+0.0197; p=0.85). C. Association between Symptoms and CFSUM1, β- alanine and CFSUM2

To assess the relationship between disease severity and metabolite concentrations the inventors compared the metabolites with symptom incidence. The concentrations of CFSUM1 and β-alanine were correlated positively with increasing symptom incidence (CFSUM1 r=+0.3223, p=0.009; β-alanine r=+0.4086, p=0.001) while CFSUM2 correlated negatively with symptom incidence (CFSUM2 r=-0.3980, p=0.001).

To independently assess the importance of each metabolite in symptom expression, the study participants (n-65) were divided into two groups based upon the concentration of CFSUM1: 1) the thirty-two subjects with CFSUM1 concentrations greater than the median

concentration; and 2) the thirty-three subjects with CFSUM1 concentrations less than or equal to the median concentration. The reporting of the core chronic disease state symptoms for each group was then assessed. Similar divisions were performed for β-alanine and CFSUM2, and the results are summarized in Table 17. No age or sex differences were found with these study subject

divisions. Apart from paraesthesia and photophobia increased concentrations of CFSUM1 were associated with increased reporting of ten core chronic disease state symptoms. β-alanine was associated with increased reporting of six core chronic disease sate symptoms but not low-grade fever of cervical lymphadenia. CFSUM2 was negatively associated with increased reporting of seven core CFS symptoms but no symptoms were associated with this metabolite alone. The association of CFSUM1 with low-grade fever and lymphadenia suggests that CFSUM1 may be of non-host origin and variations in CFSUM1, β-alanine and CFSUM2 concentrations appear important in the

variation of symptom expression.

D. Psychological status and CFSUM1, β-alanine and CFSUM2

There was an increase in the patients with SCL-90-R dimension scores ≥63 for somatization (patients with a chronic disease state-95% vs. control - 17.2%;

p<0.00001), obsessive compulsion (patients with a chronic disease state - 40% v's control - 13.3%; p<0.02),

depression (patients- 40% vs control - 15.5%; p<0.04), anxiety (patients - 30% v's control - 8.8%; p<0.03) and phobic anxiety (patients - 25% v's control - 2.2%;

p<0.003). Comparing the increase in dimension score with the metabolite concentrations by correlation statistics in all subjects revealed significant correlations between CFSUM1 an somatization (r=+0.25, p<0.05) and CFSUM2 and obsessive compulsion (r--0.248, p<0.05). No correlations were found for β-alanine. No correlations were found between CFSUM1, β-alanine or CFSUM2 and the depression, anxiety or phobic anxiety dimension scores.

III. DISCUSSION

This data shows that urinary excretion profiles are significantly different in patients having a chronic disease state compared to control subjects, which are indicative of alterations in metabolism and homeostasis. Discriminant function analysis revealed that alterations in the concentrations of CFSUM1, β-alanine and CFSUM 2 were primarily responsible for discriminating these urine profiles. CFSUM1 and β-alanine were both significantly increased in percentage abundance in the patients when compared with the control subjects whereas CFSUM2 as significantly reduced. The concentrations of these urinary products were significantly correlated with the changes observed in levels of specific urinary, organic and amino acids found altered in the patients (Table 16). In addition, the levels of CFSUM1, β-alanine and CFSUM2 were correlated with the incidence of core chronic disease state symptoms reported by study participants. Ninety five percent of the patients had increased SCL-90-R somatization dimension scores and this dimension was found to correlate with increasing CFSUM1 concentrations. The increase in the obsessive compulsion dimension was correlated negatively with the concentration of CFSUM2. No other psychological symptom changes such as depression or anxiety, detectable with the SCL-90-R were related to the discriminant metabolites. The associations between the abundances of CFSUM1, CFSUM2 and β-alanine with alterations in urinary excretion patterns and chronic disease state symptom reporting provide strong evidence for an etiological association between these metabolites and chronic disease states.

EXAMPLE 3

This example relates to analysis of the levels of CFSUM1 as well as β-alanine by multivariate analysis. CFSUM1 and /J-alanine to be the 1st (P<0.00001) and 2nd most important variables for discriminating between patients with chronic disease and control subjects. This example demonstrates an etiological relationship between CFSUM1 and/or β-alanine with chronic disease.

I. METHODS

A. Study Subjects Chronic disease patients and age and sex-matched control subjects were recruited over a six month period. The CFS group comprised patients that had previously been diagnosed as having chronic disease and met both the American CDC (Holmes et al., 1988) and the Oxford (Sharpe et al . , 1991) CFS research criteria as already reported. All subjects were provided with a questionnaire

addressing onset events, symptom incidence, pain

distribution, medical history, duration and severity of symptoms and a protocol for collection of a first morning urine sample at the first consultation. Forty five age and sex-matched control subjects were recruited from relatives of the chronic disease patients and from unrelated subjects. All samples were coded for analysis and data processing. On completion of all laboratory analyses the data were decoded and grouped for

statistical analysis. Psychological status was assessed by using the Hopkins Symptom Checklist-90-Revised

(SCL-90-R). B. symptom Indices

Symptom indices were established to assess the association between patient symptom expression and the urinary metabolites. A CFS symptom index was defined as the incidence of symptoms listed in the American CDC criteria (Holmes et al . , 1988) which included fatigue, headache, myalgia, muscle weakness, low-grade fever, sore throat, lymphodynia and photophobia. The total symptom index was defined as the incidence of all positive responses to the 48 symptoms listed in the questionnaire. Those symptoms with increased incidence in the CFS patients were then divided into infection-related, neurological, musculoskeletal, gastrointestinal and genitourinary groups with their corresponding symptoms as listed in Table 17. The respective symptom group indices were calculated as the incidence of the appropriate symptoms for each group. An index of cognitive

impairment was constructed by addition of the scalar responses to five questions from within the SCL-90-R

(Question 9. Trouble remembering things, Q38. Having to do things very slowly to ensure correctness, Q 46.

Difficulty making decisions, Q51. Your mind going blank and Q55. Trouble concentrating) (Byrne et al . , 1985). A visual analogue pain scale (VAPS) of average pain

intensity was scored as 0-4 by placement of a template over the questionnaire scale and assignment of the score. If the mark placed by the subject was coincident with a division line the subject was scored as the lower

bracket.

C. Urine Specimens and GC-MS Identification

A first morning urine specimen was collected by each subject on the day of the second visit. The urine was refrigerated (not frozen) and processed within twenty-four hours of collection. A 10 mL aliquot was centrifuged at 1,500xg for five minutes at 4ºC and a 200μL aliquot was transferred to a derivatization tube and freeze dried for eighteen hours. The freeze dried urine material was then reacted to form the

N(O,S)-heptaflurobutyryl-isobutyl (HFB-isobutyl)

derivatives for analysis by using a Hewlett Packard GC-MS (5971A mass selective detector, MSD). Twenty-eight urine peaks were selected for

retention indices and mass spectra) and the WILEY

Database^™. The peaks were numbered one to twenty-eight in order of retention time and those peaks that could not be identified were allocated a reference code (either UM or CFSUM) . The percentage abundances of the twenty-eight peaks were summarized to assess qualitative changes in urinary composition. The peak areas were not assessed against urinary creatinine or 3-methylhistidine

concentration as significant differences in metabolite, psychological inventory and symptom correlations were found between the groups in relationship to

3-methylhistidine concentration.

D. Statistical Analysis The percentage composition urine data were arcsine transformed before analysis. Subject characteristics and symptom incidence was assessed using chi-square

probability and t-tests and correction for multiplicity occurred where necessary. Symptom indices and

metabolites were compared using t-test, Mann-Whitney U test, forward stepwise multiple regression and discriminant function analyses. These data were

processed using Access^™ (Ver. 1.1, Microsoft), Excel" (Ver. 4.0, Microsoft) and Statistica^™ (Ver. 4.5,

Statsoft, Tulsa).

II. RESULTS

A. Subject Characteristics Twenty CFS [Mean age = 39.4±12.2 (sd), Range = 17-58 years, 80% female] and 45 age-and sex-matched Non-CFS controls [Mean age = 37.1±14.8 (sd), Range = 12-74 years, 71.1% female] were recruited. No significant sex or age differences were noted in any of the groups. All CFS patients complied with the Oxford and American CDC CFS symptom criteria and were consistent with a level II neurological dysfunctional grouping.

B. Symptom incidence

A questionnaire was used to survey the incidence of 48 symptoms that may potentially be associated with chronic disease patients. The analysis of these symptom incidence data by sensitivity and specificity revealed that 33 of the 48 symptoms were significantly (P<0.005) increased in the chronic disease patient group (Table 17). The symptoms were then each assessed across all 65 subjects by discriminant function analysis which

indicated that the urine profiles for subjects reporting the specific symptom were significantly different to those that did not report that symptom (model P-values in Table 17). These analyses also determined which urinary metabolite in the profile was the most important

parameter for discriminating between those subjects reporting and not reporting the symptom (summarized as the 1st discriminant metabolite in Table 17). Ten symptoms had CFSUM1 as their primary discriminant

parameter and six of these symptoms represent core CFS symptoms as described by the American CDC (Holmes et al . , 1988) . Ten symptoms had β-alanine as their primary discriminant parameter and two of these represent core CFS symptoms (Holmes et al . , 1988). Hippuric acid was the primary discriminant parameter for 2 of the 3

lymphodynia symptoms representing the remaining core CFS symptoms. CFSUM2 (chronic fatigue syndrome urinary marker -2, see Fukuda et al . , 1994), UM13 (urinary marker - 13, see Fukuda et al . , 1994), alanine and aconitic acid were primary discriminant parameters for the remaining symptoms. These symptoms could be classified into 5 major groups including infection-related, neurological, musculoskeletal, gastrointestinal and genitourinary groups and a sixth group of diverse origin.

C. Symptom Indices and Metabolite Associations Symptom indices were constructed for the

infection-related, neurological, musculoskeletal,

gastrointestinal and genitourinary groups of symptoms as well as for chronic disease symptoms, total symptoms, cognitive impairment and VAPS. The mean values for each index were calculated for the CFS patients and the non-CFS control subjects and these data are compared in Table 18. The mean index values were 3 - 11 times higher in the chronic disease patients compared to the controls and the probability statistics were all P<0.0000001.

These indices give a high specificity for symptom

expression in chronic disease patients and may therefore be used to investigate relationships between symptom expression and the corresponding urine excretion profiles in chronic disease patients and control subjects. D. Symptom Index/Metabolites Associations Assessed by Forward Stepwise Logistic Regression

Multiple regression analysis was applied to

determine whether the symptom indices were correlated with the alterations in the urine profile. All 9 symptom indices had significant associations (R²) with the changes in the urinary excretion of metabolites (P<0.0001) as shown in Table 19. Each multiple regression model had its own characteristic set of urine anomalies which are listed in the table as the primary correlates for each analysis. Increases in the relative abundance of CFSUM1 was the primary alteration in urine excretion that contributed the most in determining a high index score for the CFS core symptom, infection-related symptom and the musculoskeletal symptom indices. The relative abundance of β-alanine was the primary urinary anomaly that contributed the most in determining a high index score for the total symptom, gastrointestinal symptom and the genitourinary symptom indices. CFSUM2 was the primary correlate for the neurological symptom index and the VAPS. Hippuric acid was the prime positive

discriminant metabolite for the cognitive symptom index. CFSUM1, β-alanine and CFSUM2 were therefore strongly associated with symptom variation with hippuric acid being associated with changes in cognitive symptoms in CFS patients.

III. DISCUSSION

This Example demonstrates that excretion of CFSUM1 (amino-hydroxy-N-methyl-pyrrolidine) and β-alanine are related to the expression of the CFS core symptoms and the observed biochemical changes noted in chronic disease patients. CFSUM1 is primarily correlated with

infection-related and musculoskeletal symptoms while β-alanine is primarily correlated with the expression of gastrointestinal and genitourinary symptoms.

EXAMPLE 4

This example correlates the; 1) infective histories, 2) carriage of toxicogenic staphylococci, and 3) types of staphylococcal enterotoxins or membrane damaging toxins produced in muscle pain subjects.

I. METHODS A. Study Subjects

Forty one age/sex matched subjects were recruited from a social group over the same period and completed the study protocol. They were compared to the muscle pain group by: 1) collectively, and 2) as an asymptomatic subgroup. The asymptomatic subgroup was selected by excluding those subgroup. The asymptomatic subgroup was selected by excluding those subjects who answered

positively to any of 9 selected somatic questions in the SCL-90-R psychological inventory. The SCL-90-R somatic questions were; In the last week, how much were you distressed by: Question 1 (Q.1). Headaches; Q.12. Pains in heart or chest; Q.14. Feeling low in energy or slowed down; Q.27. pains in lower back; Q.39. Heart pounding or racing; Q.42. Muscle soreness; Q.56. Feeling weak in parts of your body; Q.58. Heavy feelings in your arms or legs; and Q.87. The idea that there is something

seriously wrong with your body. B. Questionnaire

A comprehensive questionnaire was presented to 46 sequentially presenting muscle pain subjects, over a 7 month period, who had been referred for assessment of chronic orofacial muscle pain. The questionnaire was compiled from an existing university pain clinic questionnaire, and the literature reported signs, symptoms, etiological and onset-related events for other muscle pain conditions (generalised, regional and

localised pain syndromes). The questionnaire contained 1) 4 body diagrams (left and right of head and neck, front and back of whole body to outline pain

distribution, 2) 154 questions on medical and family history, onset, exacerbation, signs and symptoms, duration, pain severity, previous treatment and potential aetiologies, and 3) a Symptom Check List-90-Revised

(SCL-90-R) psychological inventory. On completion the questionnaires were checked by the clinician and the subjects questioned in detail to confirm the answers.

Superficial muscle pain was confirmed by standard

clinical palpation methods (Helkimo, 1974). Any

medically relevant documentation that could be obtained was cited. After instruction, subject nasal and low vaginal swabs (transported in Stuarts medium) were obtained.

The nasal and genital samples were seeded onto horse blood agar and incubated at 37°C in a humidified

incubator supplemented with 10% CO₂. Staphylococcal isolates were selected by colony morphology and

identified by Gram stain morphology and standard

biochemical methods (Balows et al . , 1991; Kloos &

Schleifer, 1975).

Supernatants from staphylococcal isolates, prepared by overnight incubation in brain heart infusion broth

(BHI), were centrifuged. Membrane-damaging toxins were assessed by haemolysis assays using rabbit (α toxin), sheep (β toxin), human (δ or e toxin) and horse

erythrocytes (Mollby, 1995). Assessment of

staphylococcal enterotoxins A, B, C, D (SEA, SEB, SEC, SED) and toxic shock syndrome toxin-1 (TSST-1) was undertaken using manufactured toxin test kits (Oxoid Chemical Co.).

A toxicogenic staphylococcus was defined as one that produced >2 standard deviations from the mean percentage haemolysis on any of the erythrocyte types obtained from the asymptomatic control group.

C. statistical Analysis

Sample size assessment based upon epidemiological data suggested a minimum control group of 37 per arm, assuming a 60% incidence, whilst the asymptomatic

subgroup required a minimum of 7 per arm, assuming a 10% incidence. Sensitivity (Sen), defined as the percentage incidence of true positives in the test group, and specificity (Spec), defined as the percentage of true negatives in the comparative group, were detected.

Statistical analysis was performed using arcsin converted haemolysis percentages expressed as the mean percentage haemolysis, Chi-square (X²) and exact probability (EP) tests to compare study group incidence differences

(significance p#0.05). II. RESULTS

A. Questionnaire Results

Of the 46 muscle pain patients and 41 control subjects interviewed, three pain patients and one control subject were excluded based on incomplete questionnaires. There were no significant differences between the age and sex characteristics of the muscle pain (MP) or control (C) groups (MP: mean age 40.1±12.6 (±SD)yrs, range

16-72yrs, Female:Male ratio 3.8:1; C: mean age

34.9±14.8yrs, range l1-72yrs, Female:Male ratio 1.8:1). No significant difference was found in the number of patients or subjects in long term marital or de facto relationships (29 and 25, MP and C respectively). B. Pain Distribution

Of the 43 pain subjects, the body distribution of pain was; 1) face and head (35; 81.4%), 2)neck, shoulder and thoracic spine (38; 88.4%), 3) lower limb (30;

69.8%), 4) low back (29; 62.8%), 5) upper limb (24;

55.8%), 6) abdomen (17; 39.5%), an 7) anterior chest wall and sternum (14; 32.6%). Limb and anterior chest wall and/or sternal pain were associated with increased symptom severity. Table 20 lists the statistically increased symptoms and those that increased with severity in the pain subjects. In the male muscle pain subjects orchialgia was significantly increased (3/9 v's 0/15;

p=0.04). There were no significant differences between the groups for conditions like arthritis (11 v's 5) or chronic sinusitis (19 v's 10).

A sudden onset of symptoms was reported by 13

(30.2%) pain subjects. This sudden onset was associated with; 1) upper respiratory tract or influenza-like infection (11/13; 84.6%), 2) unexplained diarrhoea (4/13; 30.7%), 3) trauma (2/13; 15.7%), 4) a urinary tract or genital infection (3/13; 23%) and 5) glandular fever (1/13; 7.7%). A gradual onset was described by 30

(69.7%) subjects and was reported to follow; 1)

unexplained diarrhoea (6/30; 20%) , 2) an upper

respiratory or influenza-like infection (5/30; 16.7%), 3) trauma (3/30; 10%) 4) glandular fever (3/30; 10%), and 5) a urinary tract or genital infection (1/30; 3.3%).

Twelve subjects (27.9%) could not identify any

significant event at or around onset. Two subjects reported an onset-related urinary tract or genital infection after a new sexual contact. Pain at onset had highest incidence in the head, 20(46.5%), with a

facioscapulohumeral distribution being the most common, 30(69.8%). Low back pain was the first site of pain in only eight (18.6%). Localised or regional pain

progressed to generalised pain involving all four quadrants in 28(65.1%) patients.

C. Signs and symptoms

Of the 54 symptoms assessed, Table 23 lists, by sensitivity, those significantly different between the two groups. Menstrual pain, in female pain patients, was significantly increased (Sen=64.7%, Spec=72%; p=0.005) as was orchialgia in male pain patients (Sen-33.3%,

Spec=100%; p=0.04). There were no significant

differences between the groups for common conditions such as arthritis or chronic sinusitis. A group of severely affected patients, selected by the presence of widespread pain (pain in four quadrants, n=28) , were compared with the remaining muscle pain patients (regional pain group, n=15) . The widespread pain group had significantly increased incidences of limb and anterior chest wall pain (Sen=46.4%, Spec=93.4%;

p=0.007), recurrent feverishness (Sen=67.9%, Spec=80%; p=0.002), lymphadenopathy (Sen=67.9%, Spec=66.7%;

p=0.05), palpitations (Sen=67.9%, Spec=93.3%, p<0.001), nausea (Sen=92.8%, Spec=86.7%; p<0.001), irritable bowel syndrome (Sen=57. 1%, Spec=80%; p=0.01) and past and recurrent nasopharyngeal infections (Sen=64.3%,

Spec=93.3%; p<0.001). The regional pain group reported an increased incidence of bruxism (Sen=66.6%, Spec-76.6%; p=0.005) . D. Onset-related Events

A sudden onset of symptoms was reported by 13

(30.2%) MP patients. This sudden onset was associated with: 1) upper respiratory tract or influenza-like infection, 11/13(84.6%); 2) unexplained diarrhoea,

4/13(30.7%); 3) trauma, 2/13(15.7%); 4) urinary tract or genital infection, 3/13(23%); and 5) glandular fever, 1/13(7.7%). Two of the three patients with an

onset-related urinary tract or genital infection reported this to be associated with a new sexual contact. Only two of the sudden onset patients did not report an infection-related onset and both reported a

trauma-related onset event.

A gradual onset was described by 30 (69.8%) patients and was reported to follow: 1) unexplained diarrhoea, 6/30(20%); 2) an upper respiratory or influenza-like infection, 5/30(16.7%); 3) trauma, 3/30(10%); 4)

glandular fever, 3/30(10%); and 5) a urinary tract or genital infection, 1/30(3.3%). Unknown infection-like event related onset were reported by 18 of the 30

patients (60.0%). Twelve of the 30 gradual onset

patients (40%) could not identify any significant

causally-related event at or preceding onset. These represented 27.9% of the total group.

E. History of Infective Events The pain patients had significant increases in: 1) history of chronic sore throats or nasopharyngeal

infection (17 v's 2; Sen=39.5%, Spec=95%; p<0.001); 2) history of genitourinary infection (16 v's 7; Sen=37.2%, Spec=82.4%; p<0.05); 3) history of appendicitis and appendectomies (10 v's 3; Sen=23.2%, Spec-92.5%; p<0.05); and 4) reporting of increased incidence of muscle pain in their long term partners (16 v's 2; Sen=39.5%, Spec=90%; p=0.001). In the pain group a total of 29 patients were in long term relationships, of whom 17 (39.5% of the total group; 58.6% of those in a long term relationship) reported that their partner had chronic muscle pain of varying severity and distribution.

III. MICROBIOLOGY A. Total and Asymptomatic Control Groups

The asymptomatic control subgroup consisted of 9 subjects who satisfied the SCL-90-R selection criteria. There was a significant difference between the control and asymptomatic control groups in; 1) carriage of toxicogenic staphylococci (25/40 v's 1/9; p<0.005), and 2) carriage of both human (21/40 v's 0/9; p<0.001) and horse (22/40 v's 0/9; p<0.001) haemolysing

toxin-producing staphylococci. Table 21 shows the toxin production data for the 112 isolates tested (Control n-112; Asymptomatic control n=23) . The mean and 2 standard deviation range for the various

membrane-damaging toxins in the asymptomatic control group were; rabbit haemolysing - 0.23 (0-6.73), sheep haemolysing - 4.53 (0-32.93), Human haemolysing - 0.32 (0-3.01), horse haemolysing - 1.91 (0-8.47).

B. Muscle Pain Group The carriage of toxicogenic staphylococci was significantly different between the pain (37/43 or 86%) and both control (25/40) (62.5%); p<0.005) and

asymptomatic control groups (1/9 (11.1%); p<0.001).

There was no significant difference in the carriage of toxicogenic staphylococci between the control or pain subjects who responded positively to any one of the 9 somatic questions in the SCL-90-R. Table 21 and Table 22 show the toxin production of the 243 isolates tested. No significant difference existed between the carriage of SEA, SEB, SEC, SED or TSST-1 between the pain and either of the two control groups.

IV. DISCUSSION

This study has identified strong associations between chronic orofacial muscle pain and increased incidences of infective-like events, and multi-organ signs and symptoms supportive of an infective aetiology. Wider pain distribution was associated with a significant increase in incidence of multi-organ signs and symptoms supportive of an infective aetiology, such as recurrent feverishness and lymphadenopathy. These findings

indicate that orofacial pain is associated with a

systemic condition with an increased incidence of

gastro-genito-urinary symptoms and therefore represents a subcomponent of the more generalised pain syndromes such as myofascial pain and fibromyalgia syndromes (McGregor et al . , 1992).

EXAMPLE 5

This example considers the association between SCL-90-R responses and the carriage of toxicogenic staphylococci in 43 chronic muscle pain and 40 control subjects.

I. METHODS A. Subject Characteristics

A comprehensive pain questionnaire (Collaborative Pain Research Unit questionnaire; CPRU) and an SCL-90-R were presented to 46 sequentially presenting pain

patients (pain duration >3 months) and 41 healthy

age-matched controls. After detailed instruction, subjects collected nasal and in females a low vaginal swab. To allow appropriate comparison the control (C) subjects were divided into asymptomatic (AC) and

symptomatic (SC) control subgroups by their responses to 9 selected SCL-90-R somatic questions [Q1, Q12, Q14, Q27, Q39, Q42, Q56, Q58, Q87] . Similarly, the muscle pain subjects (MP) were divided into 2 groups based on the presence of widespread (WM, pain in all 4 quadrants) or regional (RM) pain. The incidence and degree of

responses to individual questions were compared between the pain and control groups (MP and C) and the subgroups (AC, SC, RM, WM) .

The microbiological data was obtained by standard methods as described in Example 1. A toxicogenic

staphylococcus was defined as one that induced haemolysis greater than 2 standard deviations from the mean

percentage haemolysis on any of the respective erythrocyte types obtained from the asymptomatic control group.

B. statistical Analysis

The haemolysis data were tested using t-test after arcsin conversion of the percentage haemolysis for each isolate tested. Sensitivity (Sen), defined as the percentage incidence of true positives in a test group, and specificity (Spec), defined as the percentage of true negatives in the comparison group, were determined.

Incidence data were compared using Chi-square and Exact probability tests (significance p 0.05). The SCL-90-R scales were compared using the t-test, cluster and discriminate function analyses to assess the changes in the dimension and index scores (Statistica (TM) , Ver.

4.3; Statsoft, Tulsa). The minimum statistical arm size for comparison of the muscle pain and asymptomatic subjects was 7.

II. RESULTS

A. Patient Characteristics Of the 46 muscle pain and 41 control subjects interviewed, 3 pain and 1 control subject were excluded on the basis of incomplete questionnaires. The control group (n=40) were divided into two SCL-90-R selected subgroups: symptomatic (SC; n=32), and asymptomatic (AC; n=8); and the muscle pain group (n=43) were divided into regional pain (RM; n-15) and widespread pain (WM; n-28) groups. The age and sex characteristics of the groups are shown in Table 24. No statistical difference was noted for any of the parameters presented. B. SCL-90-R Data

Table 25 shows the 29 SCL-90-R questions that were significantly increased in either incidence or degree of response in the muscle pain (MP) subjects compared to the control (C) subjects and divided into their

disease-related categories.

C. Toxicogenic Staphylococci and SCL-90-R

Responses

There was a significant increase in the SC, RM, and WM groups when compared with the asymptomatic (AC) control group for: 1) carriage of toxicogenic

staphylococci (p<0.001); 2) carriage of human (p<0.001) and horse (p<0.001) haemolysing toxicogenic

staphylococci; and 3) the mean percentage haemolysis by horse (p<0.001) and human (p<0.001) haemolysing toxins (see Table 26) . No significant difference was found in the incidence of carriage or haemolysin production between the symptomatic control (SC) subgroup and any of the pain (MP, RM, WM) groups.

The SCL-90-R selected asymptomatic control group did not respond to any of the significantly elevated muscle pain-related questionnaire questions and the subjects were not carriers of staphylococci producing human or horse haemolysing toxins (p<0.001). Table 27 shows the percentage carriage of toxicogenic staphylococci by positive responding subjects to the 29 muscle pain subjects related questions and comparison of incidence between muscle pain (MP) and control (C) subjects. This table clearly shows that a positive response to any of these questions is significantly related to carriage of toxicogenic staphylococci in either muscle pain (MP) and symptomatic control (SC) subjects. The 3 questions found to have significantly different levels of carriage were cognitive events. Table 28 shows the carriage data for the 9 SCL-90-R questions used to choose the asymptomatic subjects. Discriminant function analysis of the SCL-90-R dimensions with the staphylococcal haemolysis data revealed a significant effect of human haemolysing toxins upon the dimension scores across the groups (AC, SC, RM, WM; Wilks' Lambda-0.22; p=0.04). Discriminant function analysis revealed human haemolysing toxin to be the most significant discriminant factor between the asymptomatic (AS) and 1) symptomatic (SC) control (Wilks' Lambda=0.85; p=0.02); 2) regional pain (RM); and 3) the widespread pain (WM) groups. The toxin was not a significant discriminate variable in comparisons between the other groups (SC v RM or WM and RM v WM) .

III. DISCUSSION This study has: 1) demonstrated a significant association between carriage of toxicogenic staphylococci and positive replies to the 29 muscle pain associated SCL-90-R questions; 2) shown that negative responses to 9 selected SCL-90-R somatic questions could be used to select an asymptomatic control group who were not

carriers of toxicogenic staphylococci; 3) shown that there is a positive association between human and horse haemolysing toxins, the disease-related questions and the SCL-90-R index and dimension changes; 4) shown an

association exists between carriage of toxicogenic staphylococci and headaches and muscular pain and

soreness, fatigue, cognitive disturbances, sleep

disturbance, gastrointestinal and cardiac beat anomalies; and 5) shown that there is a high incidence of symptoms related to carriage of toxicogenic staphylococci within a normal control population (80%) . These findings suggest that: 1) the SCL-90-R responses are the result of a disease process; 2) toxicogenic staphylococci producing human or horse haemolysing toxins are associated with common lower grade chronic (localised and regional) pain; and 3) somatization is associated with toxicogenic staphylococcal carriage.

The lack of significant differences in toxicogenic staphylococcal carriage and toxin production between the symptomatic controls, who did not seek treatment, and the muscle pain groups, who sort treatment, suggests that other variables may influence treatment seeking. In the widespread pain (WM) subjects there were increased incidences of recurrent feverishness (p<0.001),

lymphadenopathy (p<0.001), irritable bowel (p<0.001) and symptoms of low grade diarrhoea (p<0.001), all of which suggest another etiological agent may be involved.

These data support the conclusion that the aetiology of chronic localised or regional muscle pain disorders, i.e., that pathogen induced changes are of etiological significance. In this case subjects colonised by

Staphylococcus spp, which produce lipid soluble

membrane-damaging toxins, develop a low grade toxaemia, without infection, that results in the symptoms seen in localised or regional muscle pain conditions.

EXAMPLE 6

I. METHODS A. Study subjects

CFS patients and age and sex-matched non-CFS

subjects were recruited over a six month period. The CFS group comprised patients that had previously been

diagnosed as having CFS and met both the American CDC and Oxford CFS research criteria (Holmes et al . , 1988);

Sharpe et al . , 1991). All study subjects were provided with a questionnaire addressing onset events and symptom incidence, pain distribution, medical history, duration and severity of symptoms and a protocol for the

collection of a first morning urine sample at the first consultation. Forty five age and sex-matched non-CFS control subjects were recruited from relatives of the CFS patients and from unrelated subjects. All samples were coded for analysis and data processing. On completion of all laboratory analyses, the data were decoded and grouped for statistical analysis.

B. Symptom indices

Symptom indices have been established to assess the association between patient symptom expression and the urinary metabolites (Manallack et al . , 1990). A CFS symptom index was defined as the incidence of symptoms listed in the American CDC criteria (1988) (Hyde et al . , 1992) which included fatigue, headache, myalgia, muscle weakness, low-grade fever, sore throat, lymphodynia and photophobia. The total symptom index was defined as the incidence of all positive responses to the 48 symptoms listed in the questionnaire. Those symptoms with

increased incidence in the CFS patients were then divided as infection-related, neurological, musculoskeletal, gastrointestinal and genitourinary groups with their corresponding symptoms (Manallack et al . , 1990). All these symptom indices were significantly elevated in CFS patients compared to the on-CFS controls (Manallack et al . , 1990) and were used in this study to investigate any associations with the colonisation of staphylococci.

C. Patient Microbiological Sample Collection

After detailed instruction, all subjects collected nasal swabs and the female study participants also collected low vaginal swabs. The swabs were transported in Stuart medium.

D. Staphylococcal Enterotoxin and TSST-1 Assay

The nasal and genital samples were inoculated onto horse blood agar and incubated at 37°C in a humidified incubator supplemented with 10% CO₂. Staphylococcal isolates were selected by colony morphology and coagulase was tested by the tube method (Kloos & Lambe, 1991) .

Staphylococcal isolates were grown in 5mL brain heart infusion (HHI) broth and incubated at 37ºC in a

humidified incubator supplemented with 10% CO₂ for 24 hours. The cultures were centrifuged at 2,500g at 4ºC for 20 minutes. Staphylococcal enterotoxins A, B, C, D (SEA, SEB, SEC, SED) and toxic shock syndrome toxin-l (TSST-1) were detected using thre reversed passive latex agglutination toxin test kits (SET=RPLA and TST-RPLA, respectively, Oxoid, Basingstoke). An equal volume (4μL0 of staphylococcal supernatant and either latex particles sensitized to TSST-1, SEA, SEB, SEC or SED, or a paired negative control, were mixed in a Kova slide compartment and refrigerated at 4°C for 18-24 hours. These were observed both visually and by phase contract microscopy. E. Staphylococcal Haemolysin Assay

Haemolysin were assessed by haemolysis assays with the use of rabbit, sheep, horse and human type O

erythrocytes. Supernatants from overnight growth of staphylococcal isolates in BHI broth were added to triple washed erythrocytes, suspended in 1 mM magnesium

phosphate buffered saline (0.5 KH₂PO₄ and 0.5M K₂HPO₄) to give a final concentration of 1% (v/v). An equal volume of staphylococcal supernatant was added to each of the four erythrocyte pellets and incubated at 35°C for 30 minutes. The sheep erythrocyte suspensions were further refrigerated at 4°C for 23 hours to allow for the

hot/cold haemolysis effect (Mollby, 1995). Samples were centrifuged at 3,500 for 5 minutes and the absorbance

(541 nm) was measured. The results were expressed as the percentage of the absorbance of a control tube containing totally lysed erythrocytes. The different haemolysis assays allowed detection of staphylococcal α-like

(rabbit), β-like (sheep) or tf-like toxin (human) and horse RBC haemolysin (Turner & Pickhard, 1980) . The maximum haemolysis rate per study participant was

determined by the staphylococcal isolate with the highest haemolysis rate. Haemolysis was considered significant if the value was greater than the control mean + 2 standard deviations haemolysis could be detected by the haemolysis assay.

F. Urine specimens and GC-MS identification

A first morning specimen was collected by each subject on the day of the second visit. The urine was refrigerated (not frozen) and processed within twenty-four hours of collection as already described. Urine samples were analysed using a Hewlett Packard GC-MS

(5971A mass selective detector, MSD) and twenty-eight urine peaks were identified and processed. Those peaks that could not be identified were allocated a reference code (either UM or CFSUM) (See Table 1). The percentage abundancies of the twenty-eight peaks were summarised to assess qualitative changes in urinary composition. The peak areas were not assessed against urinary creatinine or 3-methylhistidine concentration as significant

differences in metabolite correlation were found between the groups in relationship to 3-methylhistidine

concentration (Holmes et al . , 1988).

G. Statistical Analysis

The urine and haemolysis percentage data (both arcsin transformed) and clinical data were analysed using Chi-square probability, discriminant function and

multiple regression analysis (significance p≤0.05).

Correction for statistical multiplicity occurred where necessary. These data were processed using Access^™ (Ver. 2.0, Microsoft), Excel^™ (Ver. 5.0, Microsoft) and

Statistica^™ (Ver. 4.5, Statsoft, Tulsa).

II. RESULTS A. Subject Characteristics

Twenty CFS (Mean age = 39.4±12.2 (sd), Range = 17-58 years, 80% female) and 45 age- and sex-matched Non-CFS controls (Mean age = 37.1±14.8 (sd), Range = 12-74 years, 71.1% female) were selected for the study (Holmes et al . , 1988). No significant sex or age differences were noted in any other groups. All CFS patients complied with the Oxford and American CDC CFS symptom criteria and were consistent with a level III neurological dysfunctional grouping. B. incidence of staphylococcal Species,

Haemolysins and Toxins

Horse RBC haemolysin has an increased incidence in CFS patients. No enterotoxin or TSST-1 producing isolates were found in the CFS patients.

Staphylococcus aureus was found to have a reduced incidence in CFS patients. There was no difference in nasal carriage of S.aureus between males and females. There was a reduction in genital isolation of S.aureus strains compared to nasal isolation of strains in the non-CFS subjects (nose 14/63 v CFS 2.54, P<0.04) whilst no difference was noted in the CFS subjects. Similarly there was a reduction in nasal carriage of S.aureus in the CFS females compared to the non-CFS females (non-CFS 14/63 v CFS 2/54, P<0.02). All enterotoxin and TSST-1 producing isolates were nas strains of S.aureus and all were in the non-CFS subjects. S.aureus had a

significantly increased incidence of carriage per subject in the nose compared to genital carriage per subject (nasal - 21/8 v. genetical -2/54; P<0.006). Thus the major difference between CFS and non-CFS subjects, with respect to S.aureus, was the reduction of nasal carriage of S.aureus in female CFS sufferers.

S.lugdunensis and S.hominis both had increased incidence of carriage per subject in the CFS patients. No difference was noted in the incidence of S.lugdunensis or S.hominis between sexes or sampling site. Assessment of combined carriage of staphylococcal species revealed 3 patterns that only occurred within CFS patients: 1) S.lugdunensis and S.haemolyticus (4.20 v. 0/45, P<0.002); 2) S.lugdunensis and S.hominis (2/20 v 0/45, P<0.04); 3) S.hominis and S.haemolyticus (2/20 v 0/45, P<0.04).

Thus, there was an increased incidence of collective combined carriage per subject of these three staphylococci in CFS patients compared with non-CFS control subjects (8/20 v 0/45, P<0.00001).

C. Logistic Regression Assessment of

staphylococcal Species

1. Incidence of Carriage per subject.

Logistic regression analysis of the incidence of carriage per subject of staphylococcal species in CFS patients and non-CFS controls revealed a negative association with S.aureus (P<0.02) and a positive association with S. lugdunensis (P<0.02). Forward stepwise logistic regression to assess the importance of each species revealed S.aureus (-ve, P<0.02),

S. lugdunensis (+ve, P<0.03) and S.hominis (+ve, P<0.05) to be the first, second, and third staphylococcal species in determining the differences between the groups. 2. Assessment of All Isolates in each Group

Logistic regression analysis of the incidence of isolation of staphylococcal species against all isolates in CFS patients and non-CFS controls revealed a negative association with S .aureus (P<0.001) and S.warneri

(P<0.009). A positive association was revealed for

S. lugdunensis (P<0.000005), S.hominis (P<0.0001) and S.haemolyticus (P<0.0001). Forward stepwise logistic regression revealed S . lugdunensis (+ve, P<0.00004),

S.hominis (+ve, P<0.00005) and S.haemolyticus (+ve,

P<0.00003) to be the first, second, and third

discriminant staphylococcal species. D. Biochemical Changes Association with carriage of the staphylococcal Species

1. S.lugdunensis

In the S.lugdunensis positive subjects four

metabolites were increased (CFSUM1, UM17, 1- methylhistidine, UM28) and one metabolite was decreased (CFSUM2). Logistic regression analysis of species against CFSUM1 revealed a positive association between CFSUM1 and S.lugdunensis (P<0.05) and a negative

association with S.aureus (P<0.02). S.lugdunensis has an increased mean percentage haemolysis of horse RBC

haemolysin which has an increased incidence in CFS patients.

Assessment of the differences in urinary metabolites in subjects who carried S.lugdunensis in the CFS patients and the non-CFS controls revealed an increase in CFSUM2 in the CFS patients (CFS=3.03% v non-CFS=0.9%, P<0.005). There was an increase in carriage of S.haemolyticus in the S.lugdunensis positive CFS patients with none of the non-CFS subjects having joint carriage of these two staphylococci. The S.lugdunensis strains in the CFS patients differ from those in the non-CFS subjects in the production of CFSUM1 and appear to represent more

pathogenic strains.

2. S.hominis

Subjects who carried s.hominis had an increased concentration of β-alanine (S.hominis +ve 1.2±1.04% v S.hominis -ve 0.6510.66%, P<0.01). Logistic regression analysis of species against β-alanine revealed a positive association between β-alanine and S.hominis (P<0.05) whilst forward stepwise logistic regression analysis revealed S.haemolyticus (P<0.008) to be the first discriminant species for β-alanine ahead of S.hominis (P<0.04). Logistic regression analysis for metabolites against S .hominis with S.haemolyticus carriers excluded also found a significant positive relationship between S.hominis and β-alanine (P<0.02).

When all subjects who carried S.hominis were

compared with the remaining subjects they did not have ad increased carriage of S .haemolyticus . Comparison of the urinary metabolites in the CFS patients who carried

S .hominis with the non-CFS S.hominis carrier subjects revealed increases in the concentrations of CFSUM1

(P<0.005), UM13 (P<0.05) and tyrosine (P<0.05), and reductions in the concentrations of CFSUM2 (P<0.02), alanine (P<0.05) and glutamic acid (P<0.001) in the CFS patients. β-alanine is produced by bacteria via the alpha decarboxylation of aspartic acid, and enzyme not present in humans (Scriver et al . , 1978), therefore we assessed the correlations between β-alanine and aspartic acid in the subjects who carried S.hominis . No

significant relationship was found between β-alanine and aspartic acid (r*+0.2760 p-0.2768) . The reductions in the levels of alanine and glutamic acid in the CFS patients, however, support the possibility of a host based response in production of β-alanine from carnosine in muscle which normally results in alanine and glutamine release (Scriver et al . , 1978). When the CFS and non-CFS S .hominis carrier subjects were compared with the

S. lugdunensis carrier subjects excluded there was no increase in CFSUM1. This suggests that the factors associated with S .hominis pathogenicity may be related to UM13 or other as yet unidentified factors. These data suggest distinct differences exist in the S.hominis strains obtained from CFS patients and non-CFS subjects. 3. S.haemolyticus

Subjects who carried S .haemolyticus had an increased concentration of β-alanine (1.3±l.l% v 0.65±0.62%,

P<0.004) and aspartic acid (7.9% v 4.4%, P<0.006).

Logistic regression analysis of species against β-alanine revealed a positive association with S .haemolyticus

(P<0.01). There was a significant correlation between β- alanine and aspartic acid in the S .haemolyticus carrier subjects (r=+0.697, P<0.004). Thus production of β- alanine via alpha decarboxylation os aspartic acid by S.haemolyticus is very likely to be occurring (Scriver et al . , 1978). CFS S .haemolyticus positive patients have increased concentrations of CFSUM1 (P<0.04) and UM28 (P<0.04) compared with non-CFS S.haemolyticus positive subjects. However, when the CFS patients who carried S.lugdunensis were eliminated there was only an increase in aspartic acid (P<0.01) in the CFS group. Therefore, it is

unlikely that S.haemolyticus produces CFSUM1.

E. Preliminary Assessment of CFSUM1 Production by S.lugdunensis and S.hominis

As s. lugdunensis and S.hominis were found in

increased incidence in CFS patients, were found by logistic regression to be associated with CFS, were the first and second positively discriminant staphylococcal species for CFS, and were associated with increased concentrations of CFSUM1, the first discriminant

metabolite for CFS, S. lugdunensis and S.hominis were assessed for the production of CFSUM1. CFSUM2 is found in the supernatant of S.lugdunensis, implicating

S.lugdunensis in the production of CFSUM1 and the

aetiology of a chronic fatigue illness whose subjects comply with the CFS definitions. CFSUM1 was not found in the supernatants any of the S.aureus or S.hominis isolates tested to date. However, strains of S.hominis were found to produce UM13. F. Preliminary Assessment of a β-alanine

production by S .haemolyticus and S.hominis

As β-alanine correlates with aspartic acid in the subjects who carried S.haemolyticus, but not S.hominis, supernatants were tested from these organisms by CG-MS. S .haemolyticus was found to produce β-alanine whilst S .hominis did not. Thus, the microbial production of β-alanine from aspartic acid via alpha decarboxylation by S.haemolyticus is supported by these data.

G. CFS Core Symptom incidence in Subjects who

Carry S. lugdunensis, S .haemolyticus or S.hominis To determine the importance of each species in symptom expression logistic regression analysis was employed. S.hominis and S .haemolyticus against the core CFS symptoms. S. lugdunensis was found to be the most important species with highly significant relationships with the core symptoms. S.hominis. and S .haemolyticus were not significantly associated with symptom

expression. The inability to separate those species that are toxic from those that are not has place limitations on our assessment of all species, particularly S.hominis . Although combined carriage of S . lugdunensis , S .hominis, and S .haemolyticus only occurred in CFS patients the number were not sufficient to assess the results of combined carriage. H. Psychological status and S.lugdunensis,

S.haemolyticus and S.hominis

Subjects who had somatization scores >62 had an increased carriage of S. lugdunensis (>62=9/18 v <63=4/34, P<0.03) and a reduced incidence of carriage of S.aureus (>62+5/22 v <63=17/21, P<0.03). No other relationships were noted with the other dimensions or between S.hominis or S .haemolyticus and any of the SCL-90-R dimensions.

III. DISCUSSION

This study has shown that two staphylococcal

species, S. lugdunensis and S.hominis , have increased incidence whilst S.aureus, had reduced incidence in CFS patients. Subjects who carried S. lugdunensis were found to have increased urinary excretion of CFSUM1 compared to those that did not. CFSUM1 is the urinary metabolite found in increased incidence (P<0.004) and concentration (P<0.00003) in CFS patients, is statistically associated with the core CFS symptoms of CFS and was the first discriminant metabolite in a logistic regression analysis of the urinary metabolites. Supernatants of

S.lugdunensis, isolates from CFS patients were found to contain CFSUM1 which was not in the medium and therefor was produced by the bacteria. CFS subjects who had

S. lugdunensis had higher levels of CFSUM1 than non-CFS subjects who were carriers of S.lugdunensis, suggesting that certain strains of S.lugdunensis may be able to induce CFS. Two additional species, S.hominis and

S.haemolyticus , were associated with β-alanine in the urine. S .haemolyticus was found to produce β-alanine, whilst S.hominis appears associated with its production in the host as there was a reduction of glutamic acid and alanine with carriage of S.hominis and no association with aspartic acid. Interestingly, the co-colonisation of these three organisms only occurred in CFS patients. Thus, three apparently normal commensal organisms have been associated with the production of low molecular weight compounds that appear to be neurologically active metabolites and increased carriage is found in patients who report increased neurological and muscular symptoms. This is not the first time that coagulase-negative staphylococci have been shown to produce substances that alter the hosts metabolism (Bell et al . , 1991). These data therefore implicate S. lugdunensis, S .hominis , and S .haemolyticus in the aetiology of CFS.

EXAMPLE 7 I. METHODS

A. Study Subjects

As part of a clinical study on the aetiology of chronic orofacial pain, sequential pain subjects referred for assessment were examined. Twenty CFS study patients and forty-five non-CFS subjects were identified over a six month period, January to July of 1993. The CFS group comprised patients that had previously been diagnosed as having CFS and met the CFS research criteria (Holmes et al . , 1988; Sharpe et al . , 1991) as already reported. Patients and subjects were given a questionnaire

(Collaborative pain research unit - CPRU) , which was specially developed for our studies of chronic

pain/fatigue illnesses, and a SCL-90-R psychological inventory (Derogatis, 1975). All questionnaires were checked by one investigator and the patients questioned about any aspects of the questionnaire that they had trouble completing. The questionnaires, urine specimen collection and sample analysis protocol have been presented in a previous example of the present

application.

B. SCL-90-R-Inventory

The SCL-90-R has been used in epidemiological studies for CFS patients (Bode et al . , 1993) and is useful in assessing somatic and cognitive symptom severity. The study participants completed an SCL-90-R and the incidence and degree of responses to individual questions, the index and dimension scores were compared between the CFS patients and non-CFS control subjects. An SCL-90-R T-score is an age and sex normalized score which allows adequate comparison of subjects. SCL-90-R dimension T-scores greater than or equal to 63 were used to define possible psychopathological dimensions as per the SCL-90-R analysis handbook (Derogatis, 1975) .

The SCL-90-R uses the positive symptom distress index (PSDI) as an assessment of the psychological distress. In this study we defined a new index, the index of dimension T-scores ≥63, which is calculated as the number of dimensions per subject with a score ≥63. This should indicate a simple index for assessment of overall psychopathology. If CFS subjects have a form of psychological disturbance consistent with multiple elevations in dimensions as seen in major depressive illness than the PSDI and the index of dimension T-scores ≥63 should show similar results.

Urine Specimens and GC-MS Identification

The first morning urine specimens were collected by the subjects on the day of the second visit. All

subjects had been requested to cease drug, vitamin or naturopathic medicinal use for the week prior to collection. The urine was refrigerated and processed within twenty-four hours of collection. A 10 mL aliquot was centrifuged at 1,500xg for five minutes at 4ºC and a 200μL aliquot was transferred to a derivatization tube and freeze dried for eighteen hours. The freeze dried urine material was then reacted to form the N(O,S)-heptafluorobutyryl-isobutyl (HFB-isobutyl) derivatives for analysis by GC-MS. Derivatized urinary metabolites were separated using a Hewlett Packard 5890 Series II gas chromatograph (GC) and detected by a Hewlett Packard 5971A mass selector detector (MS). The data were stored and processed on a Hewlett Packard Unix-based Chemstation. The GC was fitted with a 25.0m, 0.20 mm internal diameter HP1 fused silica capillary column (film 0.33μm) and split/splitless injector. The GC-MS was run with the following settings: injector temperature 300°C, temperature program 80-300ºC at 3°C min^-1, with a 2 minute hold at 80°C and a 10 minute hold at 300°C. The mass spectrometer was set to scan from 45-650 atomic mass units every second. An injection volume of 0.5 microlitres was used.

Twenty-eight urine peaks were selected for

examination after confirming a linear detector response by the mass spectrometer. Where possible, the peaks were identified by HP-UX Chemstation computer search of user-generated reference libraries (incorporating retention indices and mass spectra) and the WILEY Database^™. The peaks were numbered one to twenty-eight in order of retention time and those peaks that could not be

identified were allocated a reference code (either UM or CFSUM) (See Table 1). The percentage abundances of the twenty-eight peaks were summarized to assess qualitative changes in urinary composition. The peak areas were not assessed against urinary creatinine or 3-methylhistidine concentration for the reasons described in a previous example.

D. Statistical Analysis

The urine data were arcsin transformed before analysis. SCL-90-R incidence data was assessed using chi-square probability, symptom indices and metabolites were compared using t-test, Mann-Whitney U test, Spearman rank order and Pearson product-moment correlations.

Forward stepwise multiple regression analysis was used to associate SCL-90-R dimension data and urinary

metabolites. These data were processed using Access^™ (Ver. 1.1, Microsoft), Excel^™ (Ver. 4.0, Microsoft) and Statistica^™ (Ver. 4.5, Statsoft, Tulsa).

II. RESULTS

A. Subject Characteristics

Twenty CFS (Mean age = 39.4112.2, Range * 17-58 years, 80% female) and 45 age- and sex-matched Non-CFS controls (Mean age = 37.1±14.8, Range = 12-74 years, 71.1% female) were recruited. No significant sex or age differences were noted in any of the groups. All CFS patients complied with the Oxford and American CDC CFS symptom criteria and were consistent with a level II neurological dysfunctional grouping. B. SCL-90-R Dimension Group Scores

Increases in all the SCL-90-R indices (global severity index - GSI; positive symptom total - PST;

positive symptom distress index - PSDI) were found in the CFS patients. The CFS patients had increased somatization, obsessive-compulsive, depression, anxiety and psychoticism dimension scores.

C. Multiple Regression Analysis of the Dimension Importance

CFS studies require the assessment of differences between subjects with depressive illness and subjects with defined CFS as both conditions may have similar clinical characteristics. Forward stepwise multiple regression analysis was used to assess which of the SCL- 90-R dimension scores were more important in determining the difference between the CFS patient group and non-CFS control group. CFS patients and non-CFS controls can be differentiated by their SCL-90-R responses (model R^o.69, F(6,58)=21.56, P<0.00001). The prime positive

discriminant dimension for determining the difference between the two groups was somatization (P<0.000009) followed by obsessive compulsion (P<0.000000005) and hostility (P=S) whilst the negative discriminant

dimensions were paranoid ideation (P<0.01), interpersonal sensitivity (P<0.002) and phobic anxiety (P=NS).

Interestingly, depression and anxiety were not found to be important intergroup determinants even though they have increased T-score responses in the CFS patients.

D. Incidence of SCL-90-R Dimension T-scores >63

1. Individual Dimension Scores >63

There was a highly significant increase in subjects with somatization T-scores >63 in the CFS patients.

Additional increases in incidence of subjects with

increased obsessive compulsion, depression, anxiety and phobic anxiety dimension T-scores ≥63 were noted when comparing the CFS and non-CFS groups. Thus the CFS group had an increased numbers of subjects who have elevated SCL-90-R dimension T-scores ≥63.

2. Multiple Dimension T-scores ≥63

There was an reduced incidence of CFS patients with no elevated SCL-90-R dimension T-scores ≥63 (CFS=1 of 20 v non-CFS=26 of 45; P<0.0001). No difference was noted in incidence of dimension T-scores ≥63 in one or 2 dimensions (CFS=11 of 20 v non-CFS=13 of 45; P=NS) however the CFS subjects had an increased incidence of subjects with somatization T-scores ≥63 (CFS*9 of 20 v non-CFS=4 of 45; P<0.0008). There was an increased incidence of CFS patients with an elevation in 3 or more dimensions with T-scores ≥63 compared with the non-CFS control subjects (CFS=8 of 20 v non-CFS=6 of 45; P<0.02). Thus the CFS group had an increased numbers of subjects who have multiple elevated SCL-90-R dimension T-scores ≥63.

E. Analysis of the Urinary Metabolite Differences Between the SCL-90-R Positive Symptom Distress Index (PSDI) and the Index of Incidence of SCL- 90-R Dimension T-scores >63

The SCL-90-R positive symptom distress index (PSDI) is an assessment of the psychological distress in the subjects assessed and was found to be increased in CFS subjects (CFS=63.4±0.9 V non-CFS=53.1±1.8, P<0.00003). The index of dimension T-scores ≥63 was also increased in CFS patients compared with non-CFS control subjects (CFS = 2.71±0.58 v non-CFS - 1.18±0.32, P<0.02).

Changes in the PSDI were associated with significant changes in urinary metabolite concentrations (Model:

R²0.430, F(13,51)=2.95, P<0.003) as was the index of dimension T-scores ≥63 (Model: R^O.362, F(10,54) =3.06, P<0.004). The PSDI had as its prime positive

discriminant metabolite CFSUM1 followed by UM13, aspartic acid, hippuric acid and phenylalanine. The index of dimension T-scores ≥63 had as its prime positive

discriminant metabolite the unknown urinary metabolite UM15 followed by hippuric acid, aspartic acid, succinic acid and β-alanine.

Thus the PSDI and the index of dimension T-scores >63 were each associated with changes in urinary

metabolite excretion however the prime discriminant metabolites were different between the two indices. Both indices shared changes in aspartic and hippuric acids and therefore had some similarities. These differences give preliminary biochemical evidence that patients with CFS differ from those with increased multiple psychological dimension scores.

F. Individual SCL-90-R Dimension and Urinary

Metabolite Associations

To further characterize the potential biochemical differences between the SCL-90-R dimensions, an

assessment of the urinary metabolite changes in subjects with dimension T-scores ≥63 was undertaken for the dimensions found to be increased in CFS patients

(somatization, obsessive compulsion, depression, anxiety, phobic anxiety). Differences were found in urinary metabolite obsessive compulsion (P<0.004), depression (P<0.0004), anxiety (P<0.0005) and phobic anxiety

(P<0.04). The prime discriminant metabolites were different for each dimension: somatization - CFSUM1;

obsessive compulsion - UM13; depression - UM15; anxiety -UM14; and phobic anxiety - alanine. CFSUM1 was not associated with changes in any dimension apart from somatization. Catecholamine based metabolites, tyrosine and phenylacetic acid, were associated with changes in the somatization, depression and anxiety dimensions.

Aspartic acid was positively associated with changes in the obsessive compulsion and phobic anxiety dimensions whilst hippuric acid was positively associated with the obsessive compulsion and depression dimensions. Lysine had a negative association with the somatization,

obsessive compulsion and depression dimensions, alanine was negatively associated with the somatization, anxiety, and phobic anxiety dimensions whilst 3-methylhistidine was negatively associated with the anxiety and phobic anxiety dimensions. These data show that each dimension is associated with excretion of different metabolites.

G. Urinary Metabolite and SCL-90-R Dimension

correlation Analysis

This analysis was used to assess which metabolites increased with increasing dimension T-scores irrespective of their clinical significance. UM15 positively

correlated with six of the nine dimensions, UM13

positively correlated with four dimensions (obsessive compulsive, depression, anxiety, psychoticism) and UM14 positively correlated with three dimensions (depression, anxiety, phobic anxiety) . Hippuric acid positively correlated with the interpersonal sensitivity and

depression dimensions. Both alanine (negative) and

CFSUM1 (positive) correlated with somatization whilst CFSUM2 negatively correlated with the obsessive

compulsive dimension. Therefore, it was found that the three unknown metabolites (UM13, UM14, UM15) were the predominant metabolites associated with changes in the SCL-90-R psychological dimensions. The metabolites found to be strongly associated with symptom expression in CFS subjects (CFSUM1, β-alanine, CFSUM2) were not strongly associated with changes in SCL-90-R psychological

dimension changes. H. Logistic Regression Associations for CFSUM1, UM13, UM14 and UM15 with the other Urinary Metabolites Strong associations were found between changes in SCL-90-R psychological inventory dimension changes and changes in urinary metabolites. This indicated that these changes may result from or be associated with separate biochemical events. To assess this, logistic regression analysis was applied to find the associations between the prime discriminant metabolites for the dimension changes (CFSUM1, UM13, UM14, UM15) with all metabolites. CFSUM1 was associated with significant changes in urinary metabolites (P<0.00001), the most prominent being the phenylalanine breakdown product, phenylacetic acid. CFSUM1 was also positively associated with the TCA cycle intermediate, succinic acid, and the muscle fibrillar protein catabolic product, 3-methylhistidine. CFSUM1 was negatively associated with CFSUM2, ethanolamine and isoleucine.

UM13 was associated with significant changes in urinary metabolites (P<0.00001), the most prominent being UM15. UM13 was also positively associated with β-alanine, proline and another unknown, UM27. UM13 was negatively associated with phenylalanine and the muscle fibrillar protein catabolic indicator, 3-methylhistidine.

UM14 was associated with significant changes in urinary metabolites (P<0.00001), the most prominent also being UM15. UM14 was positively associated with proline and UM13 and negatively associated with 3-methylhistidine, lysine and the TCA cycle intermediate, aconitic acid. UM15 was associated with significant changes in urinary metabolites (P<0.00001), the prime discriminant metabolite being UM14. UM15 was also positively

associated with UM13, phenylalanine, 3-methylhistidine and the urea cycle intermediate, ornithine. UM15 was negatively associated with proline.

CFSUM1, UM13, UM14 and UM15 were each associated with significant, yet different, changes in urinary metabolites. The changes associated with UM13, UM14 and UM15 were more alike than those seen with CFSUM1. Whilst CFSUM1 and UM15 were positively associated with the muscle protein fibrillar catabolic product, 3-methylhistidine, UM13 and UM14 were both negatively correlated with this metabolite. CFSUM1, UM13 and UM15, but not UM14, were associated with changes in

phenylalanine or its metabolite phenylacetic acid.

I. Pearson Product-moment Correlations between

CFSUM1, UM13, UM14 and UM15, and the Other

Urinary Metabolites

To further assess the associations between urinary metabolite changes and CFSUM1, UM13, UM14 and UM15,

Pearson product-moment correlation analysis was

performed.

CFSUM1 was positively associated with phenylacetic acid, succinic acid, tyrosine, aconitic acid and the unknown UM17. CFSUM1 was negatively associated with glutamic acid, CFSUM2, alanine, isoleucine, aspartic acid, ethanolamine, ornithine, proline and glycine. UM13 was positively associated with UM15, UM14, ornithine, β-alanine and proline. UM14 was positively associated with UM15, UM13, proline and isoleucine. UM15 was positively associated with UM15, UM13 and ornithine. UM13, UM14 and UM15 highly correlated with each other yet appeared related to changes in different urinary metabolites.

No relationship was found between UM13, UM14 and UM15 with previous dietary, drug, vitamin or naturopathic medicine intake.

III. DISCUSSION This study has clearly shown that CFS patients have elevated scalar responses to SCL-90-R questions in the somatization dimension and an increased incidence of patients with somatization T-scores ≥63. Nineteen of the twenty (95%) CFS patients had a somatization T-score equal to or above that required to define a somatization disorder. The CFS patients also had increased dimension T-scores ≥63 in the obsessive compulsion, depression, anxiety and phobic anxiety dimensions when compared with the non-CFS subjects. The somatization dimension was found to be primarily associated with changes in urinary amino-hydroxy-N-methyl-pyrrolidine (CFSUM1)

concentrations. CFSUM1 was found to correlate with symptom incidence and expression of the core CFS symptoms in this patient cohort and offers a biochemical basis for symptom reporting. The biochemical association between CFSUM1 and the somatization dimension therefore may exclude these patients from being defined as having somatization disorder which requires exclusion of a biochemical basis (lipowski). Similarly, the depression and anxiety dimensions, were not found to be significant dimensions in determining intergroup differences between the CFS patients and non-CFS subjects and the dimensions were also associated with unique changes in urinary metabolites. This study associates changes in SCL-90-R somatization, depression and anxiety dimensions in CFS and non-CFS control subjects with biochemically distinct urinary metabolite profiles. Furthermore this offers the possibility of a biological basis for changes in the mood disorders, depression and anxiety, as postulated by other investigators. These data do not support the hypothesis that CFS is either a somatization disorder or an

affective psychological condition.

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Claims

CLAIMS :

1. A method for diagnosing a chronic disease state comprising: obtaining a sample from an animal suspected of

having a chronic disease state; and identifying at least one microbial organism that is "present in an amount that varies from the amount observed in a healthy animal".

2. The method of claim 1, wherein said organism is selected from the group consisting of Staphylococcus spp., Candida spp., Streptococcus spp., Corynebacterium spp., Bacteroides spp., Fusiform spp., Mycobacterium spp., Clostridium spp., Proteus spp., Pseudomonas spp.. Bacillus spp., Listeria spp., Enterobacteriacaceae spp., Spirochaetes spp. and the cell wall deficient variants thereof.

3. The method of claim 1, wherein said organism is a Staphylococcus spp.

4. The method of claim 3, wherein the Staphylococcus spp. is selected from the group consisting of S . warneri , s. haemolyticus, S. xylosis, S . hominis, S. epidermidis and S. lugdunensis .

5. The method of claim 1, wherein said identifying comprises microbial culturing said sample.

6. The method of claim 1, wherein said identifying comprises measuring a bacterial product or byproduct in said sample.

7. The method of claim 6, wherein said sample is selected from the group consisting of cerebrospinal fluid, blood, urine, sputum, tears, sweat, feces or tissue.

8. The method of claim 7, wherein said sample is urine.

9. The method of claim 8, comprising the step of performing chromatographic separation of said sample.

10. The method of claim 9, wherein said measuring comprises determining the area under the peaks of a gas chromatograph profile.

11. The method of claim 10, wherein said gas

chromatograph peak is selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14, UMS15, UM15A,

UM15B, UM17 and UM28.

12. The method of claim 6, wherein said bacterial product or by-product is selected from the group

consisting of a staphylococcal α, ß, γ, δ , δ-like and horse toxin, CFSUM1 and B alanine.

13. The method of claim 1, wherein said animal is a human.

14. The method of claim 1, wherein said chronic disease state is selected from the group consisting of chronic fatigue, chronic pain, chronic inflammation, chronic depression, tinnitus, heart palpitations, migraine, short term memory loss and vision disturbances.

15. The method of claim 1, wherein said chronic pain is selected from the group consisting of muscle pain, headache, joint pain, lymph node pain, back pain and chest pain.

16. The method of claim 1, further comprising the step of performing a patient survey.

17. A method of treating a chronic disease state

comprising: identifying an animal suspected of having a chronic disease state; and providing to said animal at least one antibiotic.

18. The method of claim 17, further comprising providing to said animal at least one other composition selected from the group consisting of a toxin binding protein and a nutritional supplement.

19. The method of claim 17, wherein said antibiotic is selected from the group consisting of an antibiotic that acts on bacterial ribosomes and an antibiotic that acts on bacterial DNA.

20. A method of treating a chronic disease state comprising: identifying an animal suspected of having a chronic disease state; and providing to said animal at least one toxin binding protein.

21. The method of claim 20, further comprising providing to said animal at least one other composition selected from the group consisting of an antibiotic and a

nutritional supplement.

22. The method of claim 20, wherein said antibiotic is selected from the group consisting of an antibiotic that acts on bacterial ribosomes and an antibiotic that acts on bacterial DNA.

23. A method of treating a chronic disease state

comprising: identifying an animal suspected of having a chronic disease state; and providing to said animal at least one nutritional supplement.

24. The method of claim 23, further comprising providing to said animal at least one other composition selected from the group consisting of an antibiotic and a toxin binding protein.

25. The method of claim 24, wherein said antibiotic is selected from the group consisting of an antibiotic that acts on bacterial ribosomes and an antibiotic that acts on bacterial DNA.

26. A purified antigen composition comprising: a lipid soluble exoprotein produced by a microbial organism selected from the group consisting of Staphylococcus spp., Candida spp..

Streptococcus spp., Corynebacterium spp.,

Bacteroides spp.. Fusiform spp., Mycobacterium spp., Clostridium spp., Proteus spp.,

Pseudomonas spp.. Bacillus spp., Listeria spp., Enterobacteriaceae spp., Spirochaetes spp. and the cell wall deficient variants thereof; and a pharmaceutically acceptable carrier, buffer or

diluent.

27. A purified antigen composition comprising: an antigen selected from the group consisting of

CFSUM1, CFSUM2, UM13, UM13A, UM14, UMS15,

UM15A, UM15B, UM17 and UM28; and a pharmaceutically acceptable carrier, buffer or

diluent.

28. An antibody that recognizes an antigen selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14, UMS15, UM15A, UM15B, UM17 and UM28.

29. An antibody that recognizes a lipid soluble

exoprotein produced by a microbial organism selected from the group consisting of Staphylococcus spp., Candida spp., Streptococcus spp., Corynebacterium spp.,

Bacteroides spp., Fusiform spp., Mycobacterium spp., Clostridium spp., Proteus spp., Pseudomonas spp..

Bacillus spp., Listeria spp., Enterobacteriaceae spp., Spirochaetes spp. and the cell wall deficient variants thereof.

30. The antibody of claim 29, wherein said exoprotein is a staphylococcal α, ß, γ, δ , δ-like or horse toxin.

31. A method of generating an immune response to a chronic disease state-associated compound comprising providing to an animal an antigen composition comprising: a bacterial product or by-product; and a pharmaceutically acceptable carrier, buffer or

diluent.

32. The method of claim 31, wherein said bacterial product is a toxin or an outer membrane protein.

33. The method of claim 31, wherein said bacterial by-product is selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14, UM15, UM15A, UM15B, UM17, UM28 and ß alanine.

34. A method of preventing a chronic disease state comprising providing to an animal an antigen composition comprising: a bacterial product or by-product; and a pharmaceutically acceptable carrier, buffer or

diluent.

35. The method of claim 34, wherein said bacterial product is a toxin or an outer membrane protein.

36. The method of claim 34, wherein said bacterial by-product is selected from the group consisting of CFSUM1, CFSUM2, UM13, UM13A, UM14 , UM15, UM15A, UM15B, UM17, UM28 and B alanine.

37. A kit for use in the diagnosis of chronic disease state, in a suitable container, a first antibody that specifically binds to a microbial organism or a product or by-product thereof, the organism being present in an amount that varies from the amount observed in a healthy animal.

38. The kit of claim 37, wherein said first antibody further comprises a detectable label.

39. The kit of claim 37, wherein said kit further comprises a second antibody that specifically binds to said first antibody, the second antibody comprising a detectable label.

40. The kit of claim 37, further comprising antigen compositions, enzymes, enzyme substrate compositions and reagent for the detection of said antibody compositions.

41. The kit of claim 37, wherein said antibody is immobilized on a solid support.

42. A kit for use in the diagnosis of chronic disease state, in a suitable container, a chemical compositions that specifically derivatizes a microbial organism or a product or by-product thereof, the organism being present in an amount that varies from the amount observed in a healthy animal.

43. The kit of claim 39, further comprising reagents for the detection of said chemical compositions.