Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B5/00—Measuring for diagnostic purposes; Identification of persons
  • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
  • A61B5/411—Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/483—Physical analysis of biological material
  • G01N33/497—Physical analysis of biological material of gaseous biological material, e.g. breath

Definitions

• the present invention relates to the field of allergy tests and in particular to a method for use in the diagnosis of food allergy and food intolerance.
• Food allergies The occurrence of allergies has increased dramatically during the last decades, notably accompanied by the development of pharmaceuticals alleviating the symptoms. Although our knowledge about the causative mechanisms behind the various types of allergy has increased tremendously, all aspects are still not fully elucidated. One particularly difficult area is the area of gastrointestinal allergies, often called food allergies.
• Food allergies can be defined as the clinical symptoms resulting from an inappropriate immune response to specific food proteins or food additives.
• the broader term, food intolerance describes any adverse reaction to a food that is non-immune in nature (Burks and Stanley; Food Nllergy, Current Opinion in Pediatrics, 10(6):588-93, 1998).
• allergens have been identified and in some cases isolated and characterised down to molecular levels. In practise, however, it is often enough to identify a particular food stuff as source of allergens, e.g. cow milk containing several known allergenic proteins. Further, it is known that certain allergens often present in food, such as said cow milk proteins, hen egg albumin, fish and shell fish, nuts etc in some patients can cause an anaphylactic chock, which can be life-threatening for the patient. In other patients, the same allergens give only very weak or diffuse symptoms.
• Food intolerance is an adverse reaction to a food or food component that does not involve the IgE -mediated immune response seen in classic food allergies, e.g. cow milk allergy. Food intolerance reactions can be triggered by a physical reaction to a food or food additive. For example, people with lactose intolerance do not have enough of the enzyme lactase, to digest the lactose naturally present in milk. This is one of the most common food intolerances and can be managed through diet and the use of enzyme preparations, such as Lactaid ® caplets or drops. Another ailment is coeliac disease, where the patient reacts to the gluten in cereal foods.
• flavour enhancers mono sodium glutamate
• preservatives sulphates
• Coeliac disease or malabsorption syndrome is a syndrome mainly affecting children, but also adults, caused by a reaction to gluten which prevents the small intestine from digesting fat.
• the adverse reaction to gluten is known to cause atrophy of the intestinal villi, so that the surface available for absorption becomes smaller.
• the symptoms of coeliac disease include a swollen abdomen, diarrhoea, abdominal pains and anemia.
• Biopsies may be taken before and after exclusion of gluten in the patient's diet. Following the introduction of a gluten-free diet, the intestinal mucosa will regenerate and the symptoms normally disappear.
• FF3 Frazer's fraction III
• FF3 is a peptic-tryptic digest of gluten that is known to damage the mucosa of the small intestine in gluten-sensitive subjects.
• the biopsy samples are prepared and the number of intraepithelial lymphocytes is estimated.
• a result can be available within 24 - 48 h which prompts the authors to call this a "rapid confirmatory test of gluten sensitivity in patients already taking a gluten-free diet" (Loft, D. E. ⁇ t al, Lancet, 335 (1990) 1293-1295).
• both the SPT and the RNST tests provide useful methods for establishing whether the patient possesses IgE antibodies to specific foods.
• SPTs can be used to screen patients with suspected IgE-mediated food allergies. Glycerinated food extracts and appropriate controls are applied by the prick or skin puncture technique. Positive SPT responses indicate the possible association between the food tested and the patient's reactivity to that specific food. The positive predictive accuracy of SPTs are however less that 50 % compared with double blind placebo controlled food challenge tests (DBPCFC) according to Samson, H.N. in Food Nllergy. Part 2: Diagnosis and management (J Nllergy Clin Immunol, Vol. 103, no. 6, 1999, 981-9).
• DBPCFC double blind placebo controlled food challenge tests
• the Phadiatop test (Pharmacia & Upjohn, Inc., USA) is a multi-RNST test which gives positive result if the sera of a patient contains IgE-antibodies against one or several of the most common airborne allergens. Another test is the Phadiatop Kombi (Pharmacia & Upjohn, Inc., USA) where the above Phadiatop is combined with the RNST-FX5 test, which detects IgE-antibodies against cow milk proteins, hen egg albumin, fish, peanut, wheat and soybean.
• the ECP test (eosinophile cationic protein) measures the degree of activation of the eosinophile granulocyte, which reflects the activity of the allergic reaction in e.g. asthma and eczema.
• Examples of commercial ECP tests are UniCNPTM ECP, the Pharmacia ECP RIA and the Pharmacia CAP SystemTM ECP FEIA (Pharmacia & Upjohn, Inc., USA).
• the anamnesis remains the most important diagnostic aid, preferably supplemented with a diet diary and physical examination.
• a tentative diagnosis can be confirmed by a diet eliminating the suspected allergen and/or by oral food challenges, in sensitive patients performed under medical observation.
• U.S. 5,983,899 which method comprises analysing a sample that has been taken rectally from the large intestine of a patient who is suspected of being allergic, after having provoked the mucous membrane of the patient's large intestine rectally with an allergen against which the suspected allergy of the patient is directed.
• the method is illustrated by examples, where nine patients suffering from coeliac disease were examined using a rectal perfusion technique using a six-channel PVC hose having an inner diameter of 10 mm, an outer diameter of 16 mm and a total length of 38 cm. Three inflatable balloons were fastened to the hose, to obtain a rectal perfusion segment of 8 cm in length.
• 5,983,899 also comprises an instrument for rectal insertion in the rectum, having an expandable part which surrounds a central channel which opens out on each side of the expandable part, and a separate channel through which expansion of the expandable part can be controlled such that when the instrument is applied in the rectum; the outer wall of the expandable part will be in direct contact with the mucous membrane of the rectum; and wherein a diffusible allergen and a receptor for an inflammation marker are present on an outer defining surface of the expandable part.
• Fig. 1 shows the rectal NO concentration in seven patients following oral provocation with different allergens
• Fig. 2 shows the increase in rectal NO concentration in 18 patients, after rectal gluten provocation.
• the present inventors have surprisingly shown that the measurement of NO in the rectum or in a gas sample taken from the rectum, and in particular following provocation with the suspected allergen, is a useful tool in the diagnosis of allergy such as food allergy. It is of great importance to be able to rapidly and objectively screen patients in order to identify the individuals exhibiting a gastrointestinal allergic reaction. Regardless of the prevalence of food allergies and the notorious difficulties associated with establishing a correct diagnosis, very little advance had hitherto been made in this field.
• the method according to the present invention comprises the steps of obtaining a base line NO value for a patient, provoking the patient with the suspected allergen and obtaining a post-provocation NO value, which reflects how the patient reacts to the suspected allergen.
• the NO concentration can be measured directly in the rectum, using suitable sensors, adapted for rectal introduction, or measured in a sample, taken from the rectum. Rectal sampling results in NO values which measure both rectal and colonic reactions in case gas is allowed to freely spread in the intestinal canal.
• the sample can be taken through a catheter placed in the rectum and equipped with syringes for collecting the gas, or directly, for instance by aspirating rectal gas into a syringe.
• a sample is preferably taken by inserting an inflatable balloon, which is impermable to liquids but permeable to gases, and preferably selectively permeable to NO. When the balloon is filled with NO-free gas or with gas of a known composition, NO equilibrates over the membrane and the NO concentration in the space delimited by the membrane can be determined.
• the NO content may be determined/calculated as a concentration value, absolute amount or relative some internal or external standard.
• the content may be expressed as values normalised against components that natively are present in intestinal gas together with NO, preferably in fairly constant levels (internal standards).
• the NO content may also be calculated as amount secreted per time unit when taking air flow into account.
• sample or samples can be taken in a clinic, hospital or home environment, either by medically trained personnel or by the patient him/herself. Samples can be extracted, treated, stored and/or transported for later measurements at a different location, provided that the integrity of the sample is guaranteed or that the chemical changes taking place in the sample are known.
• NO gas analysers based on chemiluminescent detection are preferred at the priority date. See for instance Archer, FASEB J., 7 (1993) 349-60 and Alving et al, WO 9502181.
• One commercially available NO analyser is the MBA" ⁇ or NIOX"' apparatus (Aerocrine AB, Solna, Sweden).
• the NO concentration in a sample can also be determined using laser or IR based analysers or electrochemical sensors.
• the method of the present invention comprises the following steps:
• a device for measuring or monitoring NO is inserted into the rectum of a patient;
• the patient is provoked with the suspected allergen by introducing the suspected allergen in the rectum of the patient;
• the NO concentration is determined and compared to the base line value determined in a) - b), and
• an elevated value is taken as an indication of food allergy, triggered by the suspected allergen of step c).
• the method of the present invention comprises the following steps: a) a catheter having an inflatable, NO-permeable portion is inserted into the rectum of a patient;
• the inflatable portion of the catheter is inflated with NO-free gas, e.g. NO-free air, preferably using a syringe;
• the inflated catheter is kept in place for an amount of time allowing an equilibrium between the NO-concentration in the rectum and in the inflated portion of the catheter to be reached;
• a gas sample is extracted from the catheter, e.g. using a syringe
• the patient is provoked with the suspected allergen by introducing the suspected allergen in the rectum of the patient;
• the method starts by the introduction of the allergen and the value obtained after provocation is compared to compiled values representative for healthy controls; and an elevated value, compared to healthy controls, is taken as an indication of food allergy.
• an elevated value, compared to previously measured values for the same patient is taken as an indication of a worsened condition and a decreased value as an indication of an improved condition, all other circumstances being the same.
• the patient is given a suppository containing the suspected allergen or intolerance causing agent, and instructed to insert the suppository a predetermined time before the scheduled time for measurement.
• a suppository containing the suspected allergen or intolerance causing agent
• the patient is scheduled for a NO measurement in the morning, and is instructed to insert the suppository the previous evening.
• the patient is given a device for measuring and recording NO, and instructed to insert both the device and the allergen containing suppository at predetermined times, respectively.
• a diagnosis can be made.
• the diagnosis can be further confirmed by ordering the patient to follow a diet, eliminating the suspected allergen. If the suspected allergen has been effectively eliminated from the patient's diet, the NO-levels obtained at a later date should be lover than the levels initially measured and also lower than the levels measured after provocation. A repeated NO-measurement can thus help to confirm or contradict the diagnosis.
• the present invention offers an easy and repeatable method for checking or confirming, whether an allergy still exists.
• inventive method is useful for testing patient compliance.
• a patient following a diet, eliminating the source of allergen should exhibit low or normal NO concentrations in intestinal gas samples.
• High NO concentrations during ongoing elimination diet indicate either non-compliance from the side of the patient or that one or more further, unidentified allergen or allergens are present.
• step f) is preferably done with an isolated allergen or any known allergen or substance, suspected for causing an allergenic reaction in the patient to be examined.
• said substance is administered to the patient in an amount, sufficient to trigger a response in the intestinal mucosa but not so large, that a systemic response is achieved.
• This amount can be empirically determined for each allergen, groups of allergens and with respect to different patient groups, e.g. different age groups.
• the allergen is preferably administered as liquid, semi-liquid or solid composition comprising physiologically acceptable carriers and adjuvants chosen among carriers and adjuvants, normally used in pharmaceutical preparations.
• the allergen is most preferably administered in the form of a composition, suitable for rectal administration. Examples of suitable preparations include solutions, gels, pastes, suppositories and foams.
• the allergen is administered in the form of a suppository or a foam, inserted in the rectum.
• the allergen is preferably complex-bound, or physically and/or chemically encapsulated in order to limit or entirely prevent its uptake by the intestinal mucous membrane.
• the sensitivity of the determination can be increased by adding L-arginine to the preparation or by administering the substrate L-arginine perorally, intravenously, or locally e.g. via a colonoscope or a catheter to enable the detection of low grade reactions.
• the suspected intolerance causing substance is comprised in a suppository, further comprising L-arginine and suitable carriers and adjuvants, e.g. microcrystalline cellulose.
• the suspected intolerance causing substance is comprised in a foam or foam forming composition, further comprising L-arginine.
• the present invention further encompasses the use of rectal NO measurements, with or without provocation, as a method for use in screening patients exhibiting allergy related symptoms. This is based on the preliminary finding, that elevated NO values in rectally sampled air - without prior provocation - reflects systemic inflammation and - with prior provocation - local and systemic reaction to the specific agent used in the provocation. For example in very young patients with suspected atopic symptoms during the first year, a rectal NO measurement could give an indication as to the degree, character and severity of the allergy. Preliminary results also indicate, that rectal NO can be used as a predictory instrument in the examination of young patients with symptoms of allergy. High NO levels as compared to healthy individuals would indicate a disposition for the development of allergies.
• One embodiment is thus a method for use in the screening of allergic patients, exhibiting gastrointestinal problems, and where non-allergy related diseases are ruled out or held to be unlikely, according to which method the NO-concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the gastrointestinal problems of said patient.
• Another embodiment of the invention is a method for use in the screening of patients, exhibiting allergy-like symptoms in the airways, according to which method the NO- concentration is measured in a gas sample taken from the rectum of a patient and an elevated value, compared to values obtained for healthy controls, is taken as an indication of allergy as an underlying cause of the airway symptoms.
• part of the diagnosis is based on excluding other sources of NO in the intestines, e.g. inflammatory intestinal diseases.
• other sources of NO in the intestines e.g. inflammatory intestinal diseases.
• the age and condition of the patient the absence of other signs, indicating other diseases, supports the conclusion that an increased NO level is caused by allergy.
• a related application also encompassed by the present invention, concerns the differentiation between cold-related asthma and true asthma.
• a patient and in particular a young patient exhibiting breathing difficulties similar to those encountered in asthmatic patients would, according to the invention, be subjected to a rectal NO measurement.
• An elevated value, compared to healthy individuals, would be an indication of an inflammatory reaction and thus an indication of true asthma or the predisposition for the development of asthma.
• rectal NO measurement can be used to monitor the effect of treatment, e.g. steroid treatment and/or patient compliance.
• Example 1 Rectal NO levels in patients with gastrointestinal symptoms
• the inventive method was been tested on a group of young patients of both sexes (age 0.5 to 9 years, n - 5) with manifest gastrointestinal problems.
• a gas sample was obtained by inserting the tip of a conventional urological catheter into the rectum, inflating the cuff with NO-free air and extracting said air after 10 minutes.
• the NO concentration in the gas sample was measured with a Medical Breath Analyser (Aerocrine AB, Sweden). In all but two patients, the NO concentration was significantly elevated.
• the two patients exhibiting low values in parity with the NO concentrations obtained for healthy controls, were patients on an elimination diet.
• the inventive method is also useful for checking patient compliance. The results are summarised in table 1.
• the concentration of nitric oxide was determined in a gas sample taken from the rectum of patients subjected to local provocation with gluten.
• a soluble gluten extract (2 g Frazer's fraction dissolved in 10 ml saline) was introduced 15 cm into the rectum via a catheter.
• the gluten extract was kept in place 1 hour before starting the measurements.
• the concentration of NO in the rectal gas was measured by introducing NO-free air in the inflatable cuff of the catheter and waiting 10 minutes, allowing NO to penetrate into the cuff.
• the air was withdrawn from the cuff and the concentration of NO measured using a conventional NO analyser (MBA, Aerocrine AB, Danderyd, Sweden). The NO-concentration was determined at 0, 2, 4, 8 and 24 hours.
• Patient 1 51 4783 94 Patient 2 310 137 neg Patient 3 601 14000 23 Patient 4 82 152 1.8 Patient 5 870 803 neg Patient 6 22 17 neg Patient 7 36 1600 44 Patient 8 76 14000 184 Patient 9 27 750 30 Patient 10 95 273 2.9 Patient 11 119 1022 8.6 Patient 12 43 23000 535 Patient 13 32 36 1.1 Patient 14 28 8050 288 Patient 15 20 17 neg Patient 16 129 21000 163 Patient 17 60 35 neg Patient 18 8 98 12.3
• the FF3 challenge was not optimal for eliciting the right reaction in these patients. It is for example possible, that the exposure time was too short for the specific reaction pattern in these patients. Also the dose and composition of the gluten extract used in the experiments could have been less than optimal for these patients and thus be a source of error.
• the present method could make possible the differentiation between different patient groups, suffering from similar symptoms or within a group, diagnosed to suffer from coeliac disease.
• the present method could also function as a screening to distinguish between patients having symptoms, unrelated to gluten, and patients meriting a further examination.
• the inventive method has proven to be a rapid test, easy to perform and well accepted by the patients.
• the test causes no or very slight discomfort to the patient, comparable to a rectal temperature measurement.
• the method has proven to be superior to previously used in vitro tests or food challenge tests.
• Non-atopic group Controls Median 244 97 57

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