An increasing number of allergenic molecules are on the market for the goal of improving the diagnostic profile. These molecules give more information about poly-sensitizations, the distinction between co-sensitization or co-reactivity, and help to assess the potential severity of a clinical reaction, as some allergenic molecules can be ‘more dangerous’ than others. The commercially available molecules have a decision-making role within the framework of allergic immunotherapy (AIT) support and monitoring of immunological response during treatment.
by Dr F. Barocci, Dr M. De Amici, Dr S. Caimmi and Prof. G. L. Marseglia
Heterogeneity of ‘allergens’
A recombinant allergen is an allergenic molecule produced using biotechnology techniques originally identified from an allergenic extract. Recombinant allergens are produced without the proteins undergoing biological or genetic variation. This ensures consistent allergen quality, high standardization and identification of the allergenic profile of each patient, termed component resolved diagnosis (CRD) [1].
Recombinant DNA technology currently offers the possibility of producing well-defined and characterized allergens. It offers prospects of great interest from the point of view of both ‘diagnostic’ and ‘therapeutic’ avenues. The advent of recombinant allergen molecules provided new opportunities as the allergens can be produced in unlimited quantities, and innovative production techniques solve the problems concerning the cross-reactivity of IgE antibodies. Many different allergens from many different sources stimulate allergic responses from our immune system, and hence allergy diagnosis is evolving with the use of new technologies such as nanotechnologies, molecular biology, to determine ‘cross-reactivity’ and ‘co-sensitization’ [2].
Molecular-based allergy diagnostics represents a useful tool to distinguish genuine sensitizations from cross-reactions in poly-sensitized patients, where traditional diagnostic tests and clinical history are unable to identify the relevant allergens for allergen immunotherapy (AIT) [3].
AIT in an expensive treatment, typically used over longer periods of time (3 to 5 years) and correct diagnosis, selection of truly eligible patients, identification of the primary sensitizing allergen are important for optimal and cost-effective patient management.
In fact, the patient may present various positivities giving rise to a ‘poly-sensitization’, which can be differentiated into:
- ‘co-sensitization’, presence of IgE reactivity directed to distinct and structurally unrelated epitopes
- ‘co-reactivity’ (cross-reactivity), presence of IgE reactivity where IgE antibodies raised against one allergen then bind homologous molecules in a different allergen.
Allergenic molecules can be:
- ‘genuine’, specific species found exclusively in a source (food or other), indicate a real sensitization (e.g. pollen)
- ‘pan-allergens’, present in different, unrelated sources (food and non-food), indicate cross-reactivity (e.g. between food and pollen) [4].
Examples of pan-allergens are the polcalcins, allergenic calcium-binding proteins (CBPs) present in pollen of all plant species; the profilins, cytoskeletal proteins of plants present in all pollen, but also in foods of plant origin; the lipid transfer protein (LTP), present in many plant foods (particularly those in the Rosaceae family); and cross-reactive carbohydrate determinants (CCD), found in pollen, plant foods, insects and venom.
Characteristics of allergenic proteins
Allergenic proteins belong to both the Plant kingdom and the Animal kingdom, perform functions as varied as metabolic enzyme activities, structural or storage roles, some are glycosylated and some are similar structurally based on the biological relationship. The most studied and the most common allergenic molecules in the plant world are the families of proteins PR-10 (pathogenesis-related protein), known as Bet v 1 homologous proteins; the non-specific lipid transfer protein (nsLTP); profilin, also termed Bet v 2, and homologous proteins (2S albumin, 7S/11S globulin).
The vast majority (90–98%) of patients allergic to birch (family Betulaceae, order Fagales) test positive for IgE to
Bet v 1 proteins, which are thermolabile and modified during digestion [5].
The Bet v 1 specific IgE antibodies cross-react with Bet v 1 homologues present in pollen of plants included such as hazel, alder and hornbeam (family Fagaceae, order Fagales) [6] and in foods of plant origin such as apple, carrot, celery, cherry and pear. The clinical manifestations are related to the oral allergy syndrome (OAS)-type clinical reactions localized in the oral cavity and patients allergic to protein Bet v 1 homologous frequently reported good tolerance for cooked foods and commercial fruit juices.
Allergenic molecules including the birch-related profilins, or Bet v 2, are recognized in 10–20% of patients allergic to trees, grasses, herbs, fruits, vegetables, nuts, spices and latex. The Bet v 4 or calcium binding protein (CBP) allergens are present in pollen (grasses, trees, and herbs). Pollen germination occurs in the presence of calcium ions and is under the control of a class of CBPs that are found only in mature pollen. Patients who produce IgE to CBP are allergic patients or are at risk of developing allergic symptoms after contact with pollen. However, these allergens are not involved in food-plant-derived allergies.
Molecular allergens are grouped into different families depending on their molecular conformation and can provoke clinical responses of lesser (oral allergy syndrome), or greater (systemic allergic reactions) severity. The proteins PR-10 and the profilins generally are sensitive to heat and protease, so the clinical expression is related primarily to the OAS-type events. The nsLTPs and the storage proteins are not sensitive to heat or gastric digestion, and so can cause systemic reactions; however, patients allergic to LTP frequently have a good tolerance to peeled fruit [7]. Plant-based foods are a major cause of allergy and sensitivity in populations of southern Europe (Italy and Spain).
The nsLTPs are present in the Rosaceae (e.g. Pru p 3), and are also in walnut, hazelnut, corn, sesame seeds, sunflower seeds, beer, grapes, peanuts, mustard (e.g. Cor 8) [8]. The presence of LTPs in tomatoes has been highlighted, because even with peeled tomatoes, there are other LTP isoforms in the pulp and seeds [9].
The family of ‘storage proteins’ are a heterogeneous group of proteins that belong to two different superfamilies: cupins (e.g. 7/8S and 11S globulins) and prolamins (e.g. 2S albumin). The presence of IgEs against storage proteins is considered as an important marker of severe systemic reactions, for example as in allergy to peanuts (Ara h 2, Ara h 3), cereals, walnut, hazelnut, sesame, etc. These proteins are highly resistant to heat and peptic digestion and also cause sensitization in both the gastrointestinal and respiratory tracts. The substantial difference between foods of plant origin and foods of animal origin is that plant-derived foods contain both stable and labile allergenic proteins; whereas those of animal origin are mostly characterized by allergenic proteins resistant to heat and digestion [10].
The ‘opportunity’ approach
Molecular-based allergy diagnostics has emerged into routine care due to its ability to improve risk assessment, particularly for food allergies. Different foods contain unique allergenic molecules that are stable or labile to heat and digestion. The stability of a molecule and a patient’s clinical history help the clinician evaluate the risk of systemic versus local reactions. Labile allergens are linked to local reactions (typically oral symptoms) and cooked food is often tolerated, whereas stable allergens tend to be associated with systemic reactions in addition to local reactions [11].
Here, we discuss some of the most commonly used recombinant molecules for evaluating allergic patients [12].
Egg albumin
The most common of the food allergies of animal origin described here is that of egg albumen sensitivity. In this case at least two more allergens should be tested: Ovomucoid (Gal d 1) and Ovalbumin (Gal d 2) [13]. Ovomucoid is resistant to heat, urea and digestive proteases and, therefore, can trigger severe allergic reactions when the egg is ingested raw or cooked. Ovalbumin is thermo-stable, thus loses part of its allergenicity after heat treatment, and is also digested by peptidases. Ovalbumin has, then, generally lower allergenicity than ovomucoid, causing less severe allergic reactions, although occasionally exceptionally severe reactions to flu vaccines have been noted. The development of tolerance to the major molecular components of eggs is achieved normally within 4 years for ovalbumin, although not normally reached for ovomucoid. In addition, it is important to test for a reaction to egg-white lysozyme. This so-called ‘hidden’ allergen is frequently used in food preparation as a preservative and additive (e.g. in hard cheese), to prevent the formation of bacterial colonies and poses a risk to patients because it is not normally listed on food ingredient labels.
Milk
Milk contains more than 40 proteins, all of which may act as antigens for humans. Beta-lactoglobulin (BLG) and alpha-lactoalbumin (ALA) are the main proteins that are synthesized from the mammary gland, causing moderate reactions; essentially they are sensitive to heat and usually tolerance develops within 4 years. The milk of various ruminants from buffalo to cow, sheep and goat contains the same or very similar proteins that share structural and functional characteristics. Human milk contains no BLG, and the most concentrated protein is ALA, which is important in the nutrition of the newborn. Human and bovine milk differ substantially in the proportion of serum protein casein present; approximately 60 : 40 in human milk and about 20 : 80 in bovine milk and in the proportion of specific proteins. Casein is found in milk and dairy products, especially cheese, and is also often used in other foods such as sausages, soups, etc., often as a hidden ingredient. It can cause severe reactions as it is not heat labile and so tolerance does not normally develop [14].
Soybeans
One of the most important vegetables that causes allergy is soybeans. These are either used fresh or as flour, flakes, soy milk or processed to collect the oil, which is a cause of occupational asthma and is used for pharmaceuticals, cosmetics and other industrial applications. The soy allergy prevalence is estimated at 0.4% in the general population, is found in 6% of atopic children and in 14% of patients who are allergic to milk. The greatest difficulty in making a diagnosis of true soy allergy is in the differentiation of cross-reactivity with birch and peanuts [15, 16].
Shrimp
The major allergen of shrimp is tropomyosin, Pen a 1, positive in 80% of patients allergic to shellfish. It is present in muscle tissues of all living beings and therefore has a strong homology in crustaceans and shellfish (shrimp, prawns, lobster, crab, oysters, snails, squid) justifying a cross-reactivity between different species. Shrimp tropomyosin also has a high structural identity to the tropomyosin in other invertebrates, such as mites and cockroaches [17]. Patients allergic to dust mites and cockroaches will also have reactivity towards Pen a 1 without having come into contact with shellfish. Targeted immunotherapy for mite allergy can induce allergic reactions to shrimp or snails. Hence, when such therapeutic approaches are used for mite allergy, there is always the risk of causing food sensitisation in the patient.
Conclusion
Diagnostic molecular allergology is valid for discriminating allergic patients; differentiating true ‘allergies’ from ‘cross-reactivity’; leading to a more accurate ‘diagnosis’ and so reducing the need for oral food challenges; and predicting ‘severe reactions’ and ‘persistence of allergy’. Molecular diagnostics must be used for ‘targeted’ lead to a correct evaluation, and to reduce the use of oral challenges.
When a food allergen is suspected of causing allergic-type reactions of greater or lesser severity the various components of cross reactions associated with food/pollens and cross reactions between foods must be taken into account. Therefore, allergy diagnostics in vitro has often traditionally looked like positivity among individual patients giving seemingly similar laboratory results, but only the use of molecular diagnostics can draw out and highlight the differences in laboratory data in order to have a detailed specificity for various allergenic components, and then a differential clinical significance. Hence, the real situation of the patient can be defined. In order to provide the correct therapy, it is essential to know if the patient has a ‘true allergic’ reaction to the molecules specific to a particular species or if the patient has many positive results because of structural homology between different proteins.
The request for specific IgE assays should always start from a clinical evaluation and an earlier investigation in vivo or in vitro, using allergenic extracts.
References
1. Maiello N. [Allergy diagnosis: component resolved diagnosis.] Società Italiana di Immunologia e Allergologia Pediatrica, www.siaip.it (in Italian).
2. Ballmer-Weber BK, Scheurer S, Fritsche P, Enrique E, Cistero-Bahima A, Haase T, Wüthrich B. Component-resolved diagnosis with recombinant allergens in patients with cherry allergy. J Allergy Clin Immunol. 2002; 110: 167–173.
3. Alberse RC. Assessment of allergen cross-reactivity. Clin Mol Allergy 2007; 5: doi: 10.1186/1476-7961-5-2.
4. Ledesma A, Barderas R, Westritschnig K, Quiralte J, Pascual CY, Valenta R, Villalba M, Rodríguez R. A comparative analysis of the cross-reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen. Allergy 2006; 61: 477–484.
5. Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, Kraft D. IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy 1989; 44: 385–395.
6. Mari A, Wallner M, Ferreira F. Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4). Clin Exp Allergy 2003; 33: 1419–1428.
7. Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, Caldironi G. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. Allergy Clin. Immunol. 2003; 12(2): 427–432.
8. Fernández-Rivas M1, González-Mancebo E, Rodríguez-Pérez R, Benito C, Sánchez-Monge R, Salcedo G, Alonso MD, Rosado A, Tejedor MA, Vila C, Casas ML. Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population. J Allergy Clin Immunol. 2003; 112: 789–795.
9. Asero R, Mistrello G, Roncarolo D, Amato S, Caldironi G, Barocci F, Van Ree R. Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy 2002; 57(10): 900-906.
10. Van Zuuren EJ Terreehorst I, Tupker RA, Tupker RA, Hiemstra PS, Akkerdaas JH. Anaphylaxis after consuming soy products in patients with birch pollinosis. Allergy 2010; 65(10): 1348–1349.
11. Macchia D, Capretti S, Cecchi L, Colombo G, Di lorenzo G, Fassio F. Position statement: in vivo and in vitro diagnosis of food allergy in adults. It J Allergy Clin Immunol. 2011; 21: 57–72.
12. Huang F, Nowak-Węgrzyn A. Extensively heated milk and egg as oral immunotherapy. Curr Opin Allergy Clin Immunol. 2012; 12(3): 283–292.
13. Vazquez-Ortiz M, Alvaro M, Piquer M, Dominguez O, Machinena A, Martín-Mateos MA, Plaza AM. Baseline specific IgE levels are useful to predict safety of oral immunotherapy in egg-allergic children. Clin Exp Allergy 2014; 44(1): 130–141.
14. Caubet JC, Nowak-Węgrzyn A, Moshier E, Godbold J, Wang J, Sampson HA. Utility of casein-specific IgE levels in predicting reactivity to baked milk. J Allergy Clin Immunol. 2013; 131(1): 222–224.e4.
15. Kerre S. [Anaphylactic reaction to a soya dietary drink in a birch pollen allergic patient]. Revue Francaise d’Allergologie et d’Immunologie Clinique 2007; 47; 416–417 (in French).
16. Holzhauser T, Wackermann O, Ballmer-Weber BK, Bindslev-Jensen C, Scibilia J, Perono-Garoffo L, Utsumi S, Poulsen LK, Vieths S. Soybean (Glycine max) allergy in Europe: Gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy. J Allergy Clin Immunol. 2009; 123: 452–458.
17. La Grutta S, Calvani M, Bergamini M, Pucci N, Asero R. [Tropomyosin allergy: from molecular diagnosis to the clinic.] Rivista di Immunologia e Allergologia Pediatrica 2011; 2: 20–38 (in Italian).
Acknowledgement
The Authors declare no conflict of interest.
Thanks go to Cristina Torre, Giorgia Testa, Sabrina Nigrisoli for their active cooperation at the Laboratory of Immuno-Allergology, Pediatric Clinic, IRCCS Foundation Polyclinic San Matteo, Italy.
Alberto G. Martelli and Giovanni Traina, Department of Paediatrics, S. Corona Hospital, Garbagnate Milanese, Italy, are also thanked for their collaboration.
The authors
Fiorella Barocci*¹ PhD, Mara De Amici² PhD, S. Caimmi² MD, G. L. Marseglia² MD
1Department of Immunohematology and Tranfusion medicine, “di Circolo” Hospital, Rho, A.O.G Salvini Garbagnate Milanese, Italy
2Department Clinica Pediatrica, Università degli Studi di Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
*Corresponding author
E-mail: fiorellabarocci@yahoo.it
Strategies to facilitate diagnosis of allergic patients using recombinant allergens
, /in Featured Articles /by 3wmediaAn increasing number of allergenic molecules are on the market for the goal of improving the diagnostic profile. These molecules give more information about poly-sensitizations, the distinction between co-sensitization or co-reactivity, and help to assess the potential severity of a clinical reaction, as some allergenic molecules can be ‘more dangerous’ than others. The commercially available molecules have a decision-making role within the framework of allergic immunotherapy (AIT) support and monitoring of immunological response during treatment.
by Dr F. Barocci, Dr M. De Amici, Dr S. Caimmi and Prof. G. L. Marseglia
Heterogeneity of ‘allergens’
A recombinant allergen is an allergenic molecule produced using biotechnology techniques originally identified from an allergenic extract. Recombinant allergens are produced without the proteins undergoing biological or genetic variation. This ensures consistent allergen quality, high standardization and identification of the allergenic profile of each patient, termed component resolved diagnosis (CRD) [1].
Recombinant DNA technology currently offers the possibility of producing well-defined and characterized allergens. It offers prospects of great interest from the point of view of both ‘diagnostic’ and ‘therapeutic’ avenues. The advent of recombinant allergen molecules provided new opportunities as the allergens can be produced in unlimited quantities, and innovative production techniques solve the problems concerning the cross-reactivity of IgE antibodies. Many different allergens from many different sources stimulate allergic responses from our immune system, and hence allergy diagnosis is evolving with the use of new technologies such as nanotechnologies, molecular biology, to determine ‘cross-reactivity’ and ‘co-sensitization’ [2].
Molecular-based allergy diagnostics represents a useful tool to distinguish genuine sensitizations from cross-reactions in poly-sensitized patients, where traditional diagnostic tests and clinical history are unable to identify the relevant allergens for allergen immunotherapy (AIT) [3].
AIT in an expensive treatment, typically used over longer periods of time (3 to 5 years) and correct diagnosis, selection of truly eligible patients, identification of the primary sensitizing allergen are important for optimal and cost-effective patient management.
In fact, the patient may present various positivities giving rise to a ‘poly-sensitization’, which can be differentiated into:
Allergenic molecules can be:
Examples of pan-allergens are the polcalcins, allergenic calcium-binding proteins (CBPs) present in pollen of all plant species; the profilins, cytoskeletal proteins of plants present in all pollen, but also in foods of plant origin; the lipid transfer protein (LTP), present in many plant foods (particularly those in the Rosaceae family); and cross-reactive carbohydrate determinants (CCD), found in pollen, plant foods, insects and venom.
Characteristics of allergenic proteins
Allergenic proteins belong to both the Plant kingdom and the Animal kingdom, perform functions as varied as metabolic enzyme activities, structural or storage roles, some are glycosylated and some are similar structurally based on the biological relationship. The most studied and the most common allergenic molecules in the plant world are the families of proteins PR-10 (pathogenesis-related protein), known as Bet v 1 homologous proteins; the non-specific lipid transfer protein (nsLTP); profilin, also termed Bet v 2, and homologous proteins (2S albumin, 7S/11S globulin).
The vast majority (90–98%) of patients allergic to birch (family Betulaceae, order Fagales) test positive for IgE to
Bet v 1 proteins, which are thermolabile and modified during digestion [5].
The Bet v 1 specific IgE antibodies cross-react with Bet v 1 homologues present in pollen of plants included such as hazel, alder and hornbeam (family Fagaceae, order Fagales) [6] and in foods of plant origin such as apple, carrot, celery, cherry and pear. The clinical manifestations are related to the oral allergy syndrome (OAS)-type clinical reactions localized in the oral cavity and patients allergic to protein Bet v 1 homologous frequently reported good tolerance for cooked foods and commercial fruit juices.
Allergenic molecules including the birch-related profilins, or Bet v 2, are recognized in 10–20% of patients allergic to trees, grasses, herbs, fruits, vegetables, nuts, spices and latex. The Bet v 4 or calcium binding protein (CBP) allergens are present in pollen (grasses, trees, and herbs). Pollen germination occurs in the presence of calcium ions and is under the control of a class of CBPs that are found only in mature pollen. Patients who produce IgE to CBP are allergic patients or are at risk of developing allergic symptoms after contact with pollen. However, these allergens are not involved in food-plant-derived allergies.
Molecular allergens are grouped into different families depending on their molecular conformation and can provoke clinical responses of lesser (oral allergy syndrome), or greater (systemic allergic reactions) severity. The proteins PR-10 and the profilins generally are sensitive to heat and protease, so the clinical expression is related primarily to the OAS-type events. The nsLTPs and the storage proteins are not sensitive to heat or gastric digestion, and so can cause systemic reactions; however, patients allergic to LTP frequently have a good tolerance to peeled fruit [7]. Plant-based foods are a major cause of allergy and sensitivity in populations of southern Europe (Italy and Spain).
The nsLTPs are present in the Rosaceae (e.g. Pru p 3), and are also in walnut, hazelnut, corn, sesame seeds, sunflower seeds, beer, grapes, peanuts, mustard (e.g. Cor 8) [8]. The presence of LTPs in tomatoes has been highlighted, because even with peeled tomatoes, there are other LTP isoforms in the pulp and seeds [9].
The family of ‘storage proteins’ are a heterogeneous group of proteins that belong to two different superfamilies: cupins (e.g. 7/8S and 11S globulins) and prolamins (e.g. 2S albumin). The presence of IgEs against storage proteins is considered as an important marker of severe systemic reactions, for example as in allergy to peanuts (Ara h 2, Ara h 3), cereals, walnut, hazelnut, sesame, etc. These proteins are highly resistant to heat and peptic digestion and also cause sensitization in both the gastrointestinal and respiratory tracts. The substantial difference between foods of plant origin and foods of animal origin is that plant-derived foods contain both stable and labile allergenic proteins; whereas those of animal origin are mostly characterized by allergenic proteins resistant to heat and digestion [10].
The ‘opportunity’ approach
Molecular-based allergy diagnostics has emerged into routine care due to its ability to improve risk assessment, particularly for food allergies. Different foods contain unique allergenic molecules that are stable or labile to heat and digestion. The stability of a molecule and a patient’s clinical history help the clinician evaluate the risk of systemic versus local reactions. Labile allergens are linked to local reactions (typically oral symptoms) and cooked food is often tolerated, whereas stable allergens tend to be associated with systemic reactions in addition to local reactions [11].
Here, we discuss some of the most commonly used recombinant molecules for evaluating allergic patients [12].
Egg albumin
The most common of the food allergies of animal origin described here is that of egg albumen sensitivity. In this case at least two more allergens should be tested: Ovomucoid (Gal d 1) and Ovalbumin (Gal d 2) [13]. Ovomucoid is resistant to heat, urea and digestive proteases and, therefore, can trigger severe allergic reactions when the egg is ingested raw or cooked. Ovalbumin is thermo-stable, thus loses part of its allergenicity after heat treatment, and is also digested by peptidases. Ovalbumin has, then, generally lower allergenicity than ovomucoid, causing less severe allergic reactions, although occasionally exceptionally severe reactions to flu vaccines have been noted. The development of tolerance to the major molecular components of eggs is achieved normally within 4 years for ovalbumin, although not normally reached for ovomucoid. In addition, it is important to test for a reaction to egg-white lysozyme. This so-called ‘hidden’ allergen is frequently used in food preparation as a preservative and additive (e.g. in hard cheese), to prevent the formation of bacterial colonies and poses a risk to patients because it is not normally listed on food ingredient labels.
Milk
Milk contains more than 40 proteins, all of which may act as antigens for humans. Beta-lactoglobulin (BLG) and alpha-lactoalbumin (ALA) are the main proteins that are synthesized from the mammary gland, causing moderate reactions; essentially they are sensitive to heat and usually tolerance develops within 4 years. The milk of various ruminants from buffalo to cow, sheep and goat contains the same or very similar proteins that share structural and functional characteristics. Human milk contains no BLG, and the most concentrated protein is ALA, which is important in the nutrition of the newborn. Human and bovine milk differ substantially in the proportion of serum protein casein present; approximately 60 : 40 in human milk and about 20 : 80 in bovine milk and in the proportion of specific proteins. Casein is found in milk and dairy products, especially cheese, and is also often used in other foods such as sausages, soups, etc., often as a hidden ingredient. It can cause severe reactions as it is not heat labile and so tolerance does not normally develop [14].
Soybeans
One of the most important vegetables that causes allergy is soybeans. These are either used fresh or as flour, flakes, soy milk or processed to collect the oil, which is a cause of occupational asthma and is used for pharmaceuticals, cosmetics and other industrial applications. The soy allergy prevalence is estimated at 0.4% in the general population, is found in 6% of atopic children and in 14% of patients who are allergic to milk. The greatest difficulty in making a diagnosis of true soy allergy is in the differentiation of cross-reactivity with birch and peanuts [15, 16].
Shrimp
The major allergen of shrimp is tropomyosin, Pen a 1, positive in 80% of patients allergic to shellfish. It is present in muscle tissues of all living beings and therefore has a strong homology in crustaceans and shellfish (shrimp, prawns, lobster, crab, oysters, snails, squid) justifying a cross-reactivity between different species. Shrimp tropomyosin also has a high structural identity to the tropomyosin in other invertebrates, such as mites and cockroaches [17]. Patients allergic to dust mites and cockroaches will also have reactivity towards Pen a 1 without having come into contact with shellfish. Targeted immunotherapy for mite allergy can induce allergic reactions to shrimp or snails. Hence, when such therapeutic approaches are used for mite allergy, there is always the risk of causing food sensitisation in the patient.
Conclusion
Diagnostic molecular allergology is valid for discriminating allergic patients; differentiating true ‘allergies’ from ‘cross-reactivity’; leading to a more accurate ‘diagnosis’ and so reducing the need for oral food challenges; and predicting ‘severe reactions’ and ‘persistence of allergy’. Molecular diagnostics must be used for ‘targeted’ lead to a correct evaluation, and to reduce the use of oral challenges.
When a food allergen is suspected of causing allergic-type reactions of greater or lesser severity the various components of cross reactions associated with food/pollens and cross reactions between foods must be taken into account. Therefore, allergy diagnostics in vitro has often traditionally looked like positivity among individual patients giving seemingly similar laboratory results, but only the use of molecular diagnostics can draw out and highlight the differences in laboratory data in order to have a detailed specificity for various allergenic components, and then a differential clinical significance. Hence, the real situation of the patient can be defined. In order to provide the correct therapy, it is essential to know if the patient has a ‘true allergic’ reaction to the molecules specific to a particular species or if the patient has many positive results because of structural homology between different proteins.
The request for specific IgE assays should always start from a clinical evaluation and an earlier investigation in vivo or in vitro, using allergenic extracts.
References
1. Maiello N. [Allergy diagnosis: component resolved diagnosis.] Società Italiana di Immunologia e Allergologia Pediatrica, www.siaip.it (in Italian).
2. Ballmer-Weber BK, Scheurer S, Fritsche P, Enrique E, Cistero-Bahima A, Haase T, Wüthrich B. Component-resolved diagnosis with recombinant allergens in patients with cherry allergy. J Allergy Clin Immunol. 2002; 110: 167–173.
3. Alberse RC. Assessment of allergen cross-reactivity. Clin Mol Allergy 2007; 5: doi: 10.1186/1476-7961-5-2.
4. Ledesma A, Barderas R, Westritschnig K, Quiralte J, Pascual CY, Valenta R, Villalba M, Rodríguez R. A comparative analysis of the cross-reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen. Allergy 2006; 61: 477–484.
5. Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, Kraft D. IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy 1989; 44: 385–395.
6. Mari A, Wallner M, Ferreira F. Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4). Clin Exp Allergy 2003; 33: 1419–1428.
7. Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, Caldironi G. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. Allergy Clin. Immunol. 2003; 12(2): 427–432.
8. Fernández-Rivas M1, González-Mancebo E, Rodríguez-Pérez R, Benito C, Sánchez-Monge R, Salcedo G, Alonso MD, Rosado A, Tejedor MA, Vila C, Casas ML. Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population. J Allergy Clin Immunol. 2003; 112: 789–795.
9. Asero R, Mistrello G, Roncarolo D, Amato S, Caldironi G, Barocci F, Van Ree R. Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy 2002; 57(10): 900-906.
10. Van Zuuren EJ Terreehorst I, Tupker RA, Tupker RA, Hiemstra PS, Akkerdaas JH. Anaphylaxis after consuming soy products in patients with birch pollinosis. Allergy 2010; 65(10): 1348–1349.
11. Macchia D, Capretti S, Cecchi L, Colombo G, Di lorenzo G, Fassio F. Position statement: in vivo and in vitro diagnosis of food allergy in adults. It J Allergy Clin Immunol. 2011; 21: 57–72.
12. Huang F, Nowak-Węgrzyn A. Extensively heated milk and egg as oral immunotherapy. Curr Opin Allergy Clin Immunol. 2012; 12(3): 283–292.
13. Vazquez-Ortiz M, Alvaro M, Piquer M, Dominguez O, Machinena A, Martín-Mateos MA, Plaza AM. Baseline specific IgE levels are useful to predict safety of oral immunotherapy in egg-allergic children. Clin Exp Allergy 2014; 44(1): 130–141.
14. Caubet JC, Nowak-Węgrzyn A, Moshier E, Godbold J, Wang J, Sampson HA. Utility of casein-specific IgE levels in predicting reactivity to baked milk. J Allergy Clin Immunol. 2013; 131(1): 222–224.e4.
15. Kerre S. [Anaphylactic reaction to a soya dietary drink in a birch pollen allergic patient]. Revue Francaise d’Allergologie et d’Immunologie Clinique 2007; 47; 416–417 (in French).
16. Holzhauser T, Wackermann O, Ballmer-Weber BK, Bindslev-Jensen C, Scibilia J, Perono-Garoffo L, Utsumi S, Poulsen LK, Vieths S. Soybean (Glycine max) allergy in Europe: Gly m 5 (beta-conglycinin) and Gly m 6 (glycinin) are potential diagnostic markers for severe allergic reactions to soy. J Allergy Clin Immunol. 2009; 123: 452–458.
17. La Grutta S, Calvani M, Bergamini M, Pucci N, Asero R. [Tropomyosin allergy: from molecular diagnosis to the clinic.] Rivista di Immunologia e Allergologia Pediatrica 2011; 2: 20–38 (in Italian).
Acknowledgement
The Authors declare no conflict of interest.
Thanks go to Cristina Torre, Giorgia Testa, Sabrina Nigrisoli for their active cooperation at the Laboratory of Immuno-Allergology, Pediatric Clinic, IRCCS Foundation Polyclinic San Matteo, Italy.
Alberto G. Martelli and Giovanni Traina, Department of Paediatrics, S. Corona Hospital, Garbagnate Milanese, Italy, are also thanked for their collaboration.
The authors
Fiorella Barocci*¹ PhD, Mara De Amici² PhD, S. Caimmi² MD, G. L. Marseglia² MD
1Department of Immunohematology and Tranfusion medicine, “di Circolo” Hospital, Rho, A.O.G Salvini Garbagnate Milanese, Italy
2Department Clinica Pediatrica, Università degli Studi di Pavia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
*Corresponding author
E-mail: fiorellabarocci@yahoo.it
Precise diagnosis of allergies by multiplex profiling
, /in Autoimmunity & Allergy, Featured Articles /by 3wmediaby Dr Jacqueline Gosink Differential allergy diagnostics using molecular components is a powerful tool for pinpointing the precise trigger of an allergy, enabling targeted immunotherapy and comprehensive risk assessment. Multiparameter systems streamline the diagnostic procedure by delivering a comprehensive and detailed patient profile in a single test. The multiplex EUROLINE DPA-Dx (defined partial allergen diagnostics) […]
Molecular differentiation of ulcerative colitis and Crohn’s colitis: is it achievable?
, /in Featured Articles /by 3wmediaDifferentiating ulcerative colitis from Crohn’s colitis among patients with indeterminate colitis (IC) is a major challenge. The definitive diseases share demographic and clinical features, yet differ in tissue inflammation and damage suggesting distinct mechanisms. Since treatments differ, a molecular diagnostic from accessible clinical samples would greatly benefit IC patients.
by Amanda Williams and Dr Amosy M’Koma
Background
Predominantly, colonic inflammatory bowel disease (IBD), or the colitides, encompasses ulcerative colitis (UC) and Crohn’s colitis (CC) [1, 2], and (when state-of-the-art diagnostic criteria for either are inconclusive) indeterminate colitis (IC) [3]. UC and CC share many demographic and clinical features yet present significant differences in tissue inflammation and damage, suggesting a distinct etiopathogenic trigger [4]. It is believed theoretically that IBD is caused by inappropriate activation of the mucosal immune system against commensal bacteria in the intestinal lumen [4]. Differentiating UC and CC among patients with IC has remained a major challenge in endoscopic precision medicine [5]. Disease unpredictability, treatment side-effects, potential surgery, interim morbidity and acute incapacitation are individual and system burdens [6]. Because treatments for the two diseases are different, identifying phenotype-specific molecular markers would be invaluable for developing diagnostic and prognostic tools, and for precise treatment [7–9].
The need for IC classification into UC and CC is urgent for patients suffering from IBD [10]. Patients diagnosed with IC are young [11], with onset of symptoms before or shortly after the age of 18 years [11, 12] and have an equal gender distribution [13]. This contrasts to UC where there is a male predominance and a mean age of onset at 36–39 years [14]. These figures have persisted despite the introduction of newer diagnostic modalities [15]. Even after long-term follow-up, a substantial number of patients with IC still retain the diagnosis [15]. The continued presence of an IC diagnosis over time supports part of our hypothesis that IBD may represent a spectrum of diseases rather than just two the entities of CC and UC. In order to understand and resolve this challenge, an exclusion tool for differential diagnosis is needed.
To date there is no diagnostic gold standard tool for IBD. Clinicians use an inexact classification system which combines clinical, endoscopic, radiological, and histopathological techniques in order to diagnose CC and UC [15]. Even with a combination of these methods, IBD patients are mistakenly diagnosed 30% of the time [15], resulting in inappropriate pharmacologic and surgical interventions, with correspondingly significant complications [16]. The most difficult and painstaking post-operative experience is when patients pouch-operated for definitive UC change in their diagnosis to de novo Crohn’s ileitis (CI) of the ileal pouch [15]. Currently, little is known about the molecular differences distinguishing UC and CC [7, 8]. Trends in the IBD field focus on genetic susceptibility, role of normal flora, inflammatory processes, and interactions between normal flora and the immune response [17]. Even though current research is promising [8, 15], there have been no definitive answers to help clinicians differentiate between the two diseases when current diagnostics prove inadequate and result in a diagnosis of IC [3]. Rising incidence and prevalence of IBD (Fig. 1) across the world [18] is accompanied by an increase in cases of IC [11, 19]. It is becoming even more important to find molecular markers of disease to distinguish between CC and UC in patients with IC [7, 8].
Transcriptome analysis
Recently, we have quantitated the global expression profiles of RNA levels using oligonucleotide microarray/genome-wide transcriptome analysis [20, 21] to investigate transcriptional signatures present in colonic tissues obtained from UC and CC mucosa and submucosa. We used genomic data mining from pragmatic studies to demonstrate how biomedical studies can use the technology. By extracting new and useful biomedical knowledge, we hope to develop significant momentum for applications that may have medical diagnostic potential in IBD laboratories. The genomic patterns we noted show greater intensity in CC versus UC, perhaps indicative of a greater degree or different type of inflammation in the tissues underlying the layers [8]. It is possible also that these differing genes may represent candidate biomarkers that could delineate the inflammatory colitides. Specifically, these genes were noted to show greater intensity in the CC submucosa, perhaps indicative of the greater degree or different type of inflammation in the underlying tissue [20, 21]. These studies identified genes involved in inflammatory responses generally overexpressed in IBD and demonstrate that the colonic tissue transcriptomes obtained from UC/CC patients were quite different. The gene sets identified appear to distinguish UC from CC, and may serve as an excellent resource for professionals involved with gene expression data mining in a variety of clinical settings (Table 1).
Proteomics
More recently, we have developed a proteomic approach to delineating UC versus CC. Using histologic mucosal and submucosal tissue layers for analyses, we used MALDI MS for proteomic profiling along with bioinformatics technologies (Fig. 2) [7, 8]. We profiled surgical pathology resections of colonic mucosal and submucosal layers of patients with IBD undergoing colectomy in connection with pouch surgery [restorative proctocolectomy (RPC) and ileal pouch-anal anastomosis (IPAA)] [7, 8, 21]. We identified and compared protein profiles which had the necessary: (1) specificity; (2) sensitivity; (3) discrimination; and (4) predictive capacity to determine the heterogeneity of IBD7, and we were able to delineate UC and CC molecularly [7]. These molecular fingerprints are independent of tissue (mucosa, submucosa, or both) and appear to represent disease-specific markers (Table 1) [7]. Once these markers are further tested, we can potentially develop IBD screening tools which will rely on antibodies to the protein(s) of interest (Fig. 3). The distinction between UC and CC is of the utmost importance when determining candidacy for a pouch surgery [22–24]. Approximately 30% of IBD patients [7] face potential morbidity from an incorrect diagnosis with consequently inappropriate and unnecessary operative surgeries, underscoring the necessity of research efforts aimed at a more accurate diagnosis of the colitides [7, 20].
Peripheral blood biomarkers
In contrast to colon surgical pathology tissue resections, peripheral blood is a much more accessible source of cells that might be used to distinguish between CC and UC. Circulating peripheral blood cytokines are responsible for surveying the body for signs of disease. Cytokines may, therefore, serve as surrogates for disease-induced gene expression as biomarkers of disease status or severity. In pursuit of this, we studied differences in the serum cytokine behaviours between UC and CC patients [9]. We aimed so that, if successful, such analysis could lead to an assay that could be applied as an easy, accurate, affordable, non-invasive and fast screening test. However, although certain cytokines were found to differ between diseases and controls, no cytokine could clearly distinguish UC from CC [9]. An analysis of the literature has shown that although several attempts have been made to define the serum cytokines profile in IBD, the contradictory results of these studies do not indicate the possibility of finding the biomarker(s) among the serum cytokines at this time.
Differential diagnosis and treatment
These studies are highly relevant for creating a molecular differentiator for IC. Curative treatment for UC is often surgical, involving RPC and IPAA [6, 22]. Successful surgery removes the entire diseased colon while preserving bowel evacuation, continence and fertility [22]. This is largely a result of careful patient selection combined with meticulous surgical technique, but most importantly correct diagnosis [16, 22]. Clinical observations and experience suggest that it is difficult to identify patients with CC who are likely to have a successful outcome after RPC and IPAA surgery [6, 16, 23]. Thus, pouch surgery should be widely contraindicated by CC, but be an acceptable intervention for patients with UC and for those with IC who are likely to develop UC.
Despite the increased use of cutting-edge technologies, there is no single, straight- forward explanation for the heterogeneous results, and current approaches still require validation, and subsequently confirmation on patient outcomes in a large-scale clinical cohort.
Conclusion
Our multilevel transcript observations by proteomics and genomics in tissue and blood suggest that the development of a molecular biometric-based tool that can complement the inexact classification system for diagnosis of UC and CC with precision in IBD is still preliminary.
References
1. M’Koma AE, et al. Annual Congress – Digestive Disease Week, Chicago, IL, 2009; M1096 P600
2. Burakoff R. J Clin Gastroenterol. 2004; 38: S41–43.
3. Ballard BR, et al. World J Gastrointest Endos. 2015; 7: 670–674.
4. Podolsky DK. N Engl J Med. 2002; 347: 417–29.
5. North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition, et al. J Pediat Gastroenterol Nutr. 2007; 44: 653–674.
6. Keighley MR. Acta Chir Iugosl. 2000; 47: 27–31.
7. M’Koma AE, et al. Inflamm Bowel Dis. 2011; 17: 875–883.
8. Seeley EH, et al. Proteomics Clin Appl. 2013; 7: 541–549.
9. Korolkova OY, et al. Clin Med Insingts Gastroenterol. 2015: 8: 29–44.
10. Telakis ET. Ann Gastroenterol. 2008; 3: 173–179.
11. Malaty HM, et al. J Pediat Gastroenterol Nutr. 2010; 50: 27–31.
12. Kugathasan S, et al. J Pediatrics 2003; 143: 525–531.
13. Lindberg E, et al. J Pediat Gastroenterol Nutr. 2000; 30: 259–264.
14. Lee KS, et al. Arch Pathol Lab Med. 1979; 103: 173–176.
15. M’Koma AE. World J Gastrointest Surg. 2014; 6: 208–219.
16. Shen B. Inflamm Bowel Dis. 2009; 15: 284–294.
17. Corfield AP, et al. Bioch Soc Trans. 2011; 39: 1057–1060.
18. M’Koma AE. Clin Med Insights Gastroenterol. 2013; 6: 33–47.
19. Malaty HM, et al. Clin Exp Gastroenterol. 2013; 6: 115–121.
20. M’Koma A, et al. Gastroenterology 2010; 138: S-525.
21. M’Koma AE, et al. Oral presentation at the annual congress of The American Society of Colon and Rectal Surgeons, Minneapolis, MN, USA 2010: 117.
22. M’Koma AE, et al. Int J Colorectal Dis. 2007; 22: 1143–1163.
23. Shen B, et al. Inflamm Bowel Dis 2008;14:942–948.
24. Shen B, et al. Clin gastroenterol Hepatol. 2008; 6: 145–158.
The authors
Amanda Williams1 MS; Amosy M’Koma*2,3,4 MD, PhD
1School of Medicine, Meharry Medical College, Nashville, TN, USA
2Department of Biochemistry and Cancer Biology, School of Medicine, Meharry Medical College, Nashville, TN, USA
3Department of Surgery, Vanderbilt University School of Medicine, Nashville, TN, USA
4Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA
*Corresponding author
E-mail: amkoma@mmc.edu
Measuring infliximab and adalimumab drug and antibodies in Crohn’s disease and ulcerative colitis
, /in Featured Articles /by 3wmediaThe anti-TNF therapies infliximab and adalimumab have revolutionized the treatment of inflammatory bowel disease, being very effective in many patients. Some patients experience problems such as loss of response, which is associated with production of antibodies to the therapy. Measuring trough drug and antibody concentrations may direct patient management in future.
by Dr Mandy Perry, Dr Tim McDonald, Adrian Cudmore, Dr Tariq Ahmad
Ulcerative colitis (UC) and Crohn’s disease (CD) are relapsing and remitting inflammatory disorders of the gastrointestinal (GI) tract. Recently published data suggests that as many as 620 000 people in the UK could have these inflammatory bowel diseases (IBDs). Both conditions can produce symptoms of urgent and frequent diarrhea, rectal bleeding, pain, profound fatigue and malaise. In some patients, there is an associated inflammation of the joints, skin, liver or eyes. Malnutrition and weight loss are common, particularly in CD. These conditions can cause considerable disruption to education, working, social and family life. There is currently no cure. Drugs to suppress the immune system are the mainstay of medical management, and first line treatment typically includes corticosteroids, with immunmodulators such as azathioprine, mercaptopurine or methotrexate used for patients with steroid-dependent disease. However, 30% of patients either fail to respond, or are intolerant, to these drugs and will then be considered for biological therapies or surgery. More than half of patients with CD and about 20–30% of patients with UC will require surgery at some point. The anti-TNF agents infliximab and adalimumab have revolutionized treatment of IBD, and are an effective alternative to surgery, leading to complete remission in many patients [1].
NICE has published guidelines for the use of anti-TNF agents for CD [2] and UC [3]. These drugs include the monoclonal anti-TNF drugs infliximab (includes the original product – Remicade, and biosimilar infliximab Remsima and Inflectra) and adalimumab (Humira). TNF is a cytokine involved in systemic inflammation and the anti-TNF drugs bind to and inactivate TNF, thereby halting the immune cascade and reducing inflammation. Infliximab is a mouse–human chimeric anti-human TNF antibody which is administered by intravenous infusion with a typical induction course of therapy at weeks 0, 2, 6 and then 8-weekly maintenance dose. Adalimumab is a fully human anti-human TNF antibody, and is administered by subcutaneous injection every 2 weeks. For some patients this is a more convenient option, as the subcutaneous rather than intravenous administration means that frequent hospital appointments are not required. Both infliximab and adalimumab are expensive treatments, typically costing in excess of £10,000 per annum. The 2015 introduction of biosimilar infliximab preparations has significantly reduced the price of therapy.
Some patients have an excellent response to anti-TNF treatment, managing to obtain complete remission of CD and mucosal healing. However, a proportion of patients do not respond well to anti-TNF therapy [4], and there are three principal problems:
The etiology of these problems is unknown, although the following causes have been indicated [1]:
Approximately 25% of patients will develop antibodies to infliximab and adalimumab drugs within 12 months of treatment initiation. The clinical importance of such antibodies is not completely understood. It is hypothesized that anti-drug antibodies may alter the action of the drug (i.e. neutralizing) and/or increase the drug clearance (i.e. non-neutralizing). Antibodies may be transient (and may be ‘overcome’ by increasing the concentration of drug), or persistent (and intolerant of drug escalation) [6, 7].
Measuring drug and anti-drug antibodies may enable problems such as ADR, PNR and LOR to be further understood, and may assist clinicians in the management of these problems. Possible interventions include escalating the dose of anti-TNF therapy, adding in an additional drug (e.g. immunomodulator or steroid), switching to an alternative anti-TNF therapy or switching to a non-TNF biologic.
For infliximab, several algorithms for patient management have been developed using drug and antibody levels [8, 9]. Several different assays, using different therapeutic ranges have been employed as part of these algorithms, making comparison difficult. The widely quoted TAXIT (Trough level Adapted infliXImab Treatment) trial uses a therapeutic range for infliximab of 3–7 mg/L [8], whereas work by Steenholdt uses 5–10 mg/L [9]. The different technologies used to measure drug and anti-drug antibodies, include ELISA (enzyme-linked immunosorbent assay), HMSA (homogeneous mobility shift assay) [10], radioimmunoassay, and a functional cell-based reporter gene assay [11]. There is poor agreement between the drug assays, as there is neither gold standard material, nor a reference method available.
Clinicians and laboratories should also be aware that there is considerable variation in what is being measured for the antibody assays. For example, the ELISA antibody assays either measure free (i.e. only those antibodies which are not bound to drug in the patient serum) or total antibodies (i.e. bound and unbound to drug). The functional cell-based assay is different again, as it is designed to detect only those antibodies which prevent infliximab from binding to TNF and therefore may not detect antibodies that are postulated to alter the drug clearance only. Although anti-TNF drug and antibody testing shows promise, there is not yet sufficient cost-effective data, nor diagnostic algorithms, for widespread adoption across the NHS. It seems likely that use in the setting of loss of response will enter clinical practice first and may allow cost savings by avoiding dose escalation in patients with high levels of antibodies.
The Personalized Anti-TNF Therapy in Crohn’s disease (PANTS) study is a prospective, observational study for which anti-TNF naïve patients aged 6 and over are eligible. The study aims to investigation the clinical, serological and genetic factors that determine PNR, LOR and ADR to anti-TNF drugs in patients with active luminal Crohn’s disease. The study is recruiting from over 110 UK hospitals currently participating in the UK Inflammatory Bowel Disease Genetics Consortium pharmacogenetic programme. While attending routine clinical appointments, additional information and samples are collected for the PANTS project. This includes the Harvey Bradshaw index (HBI, a scoring system which classifies recent disease in terms of symptoms), blood for DNA, RNA, CRP (C-reactive protein), anti-TNF drug and antibody levels and stool samples for calprotectin. Analysis of CRP, calprotectin and anti-TNF alpha drug and antibody levels is undertaken at the Central Laboratory at Exeter Blood Sciences Laboratory, where a biobank of additional serum aliquots is being constructed. Infliximab and adalimumab drug levels, total anti-infliximab antibody and total anti-adalimumab antibody are measured by ELISA technology (Immundiagnostik), using a liquid handling robot (DS2, DYNEX Technologies) [12]. Biochemical data is uploaded onto a bespoke web-based database that is also used to store the clinical information.
Examples of data for two patients from the PANTS study are shown in Table 1. Table 1A is data from a patient who is prescribed infliximab. Week 0 shows baseline data before treatment with infliximab. The calprotectin is raised, indicating active inflammation, and this is mirrored with the CRP and Harvey Bradshaw Index (HBI score of <5 indicates remission; 5–7 mild disease, 8–16 moderate disease and >16 severe disease). By week 14, the calprotectin has decreased substantially, and the CRP and HBI have decreased to normal values at week 2. Until the end of the timeframe (week 126), the patient continues to have a normal calprotectin, CRP and HBI. The drug level concentration in the maintenance phase is between 3–14 mg/L and the patient remains negative for anti-drug antibodies (i.e. <10 AU/mL). Table 1B shows data from a pediatric patient who is prescribed infliximab. The PCDAI (Pediatric Crohn’s Disease Activity Index) is used in place of the HBI. When infliximab naïve (week 0), the patient had a raised CRP, calprotectin and PCDAI (<10 remission; 10–29 mild disease; 30–39 moderate disease; >40 severe disease). Upon treatment with infliximab there was initially a good response, shown by the decrease in CRP and PCDAI. At week 22, the patient became positive for anti-drug antibodies and the trough drug concentration became undetectable. At week 26, the patient had clinical loss of response and underwent surgery. Knowledge of the patient’s drug and antibody levels helps with clinical management in the setting of loss of response, such as in this case. Dose escalation is likely to be futile and costly in patients with high antibody titres. Switching to an alternative anti-TNF might provide transient benefit, although patients who form antibodies to one anti-TNF are likely to form antibodies to the second and subsequent agents in this class.
The anti-TNF drugs infliximab and adalimumab are effective treatment for CD in many patients. However, LOR, PNR and ADR are significant problems, and it is so far unclear as to how these patients should be best managed. Measuring drug and antibody concentrations may allow for diagnostic algorithms to be produced. The clinical and cost effectiveness of therapeutic monitoring of TNF inhibitors using ELISA technology is currently being evaluated by NICE [13]. It is anticipated that data from the PANTS study will directly inform such algorithms and guidelines, and contribute to an evidence based medicine approach for management of CD patients who are prescribed anti-TNF therapy.
References
1. Vande Casteele N, Feagan BG, Gils A, et al. Therapeutic drug monitoring in inflammatory bowel disease: current state and future perspectives. Curr Gastroenterol Rep. 2014; 16: 378.
2. NICE technology appraisal guidance [TA187]. Infliximab (review) and adalimumab for the treatment of Crohn’s disease. NICE 2010. (https://www.nice.org.uk/guidance/ta187/chapter/1-guidance).
3. NICE technology appraisal guidance [TA329]. Infliximab, adalimumab and golimumab for treating moderately to severely active ulcerative colitis after the failure of conventional therapy (including a review of TA140 and TA262). NICE 2015. (https://www.nice.org.uk/guidance/ta329)
4. Nielsen OH, Seidelin JB, Munck LK, Rogler G. Use of biological molecules in the treatment of inflammatory bowel disease. J Int Med. 2011; 270: 15–28.
5. Vande Casteele N, Ballet V, Van Assche G, et al. Early serial trough and antidrug antibody level measurements predict clinical outcome of infliximab and adalimumab treatment. Gut 2012; 61: 321.
6. Hanauer S, Feagan B, Lichtenstein G, et al. Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet 2002; 359: 1541–1549.
7. Cornillie F, Hanauer B, Diamond R, et al. Postinduction serum infliximab trough level and decrease of C-reactive protein level are associated with durable sustained response to infliximab: a retrospective analysis of the ACCENT I trial. Gut 2014; 63; 1721–1727.
8. Vande Casteele N, Ferrante M, Van Assche G, et al. Trough concentrations of infliximab guide dosing for patients with inflammatory bowel disease. Gastroenterology 2015; 148: 1320-1329.
9. Steenholdt C, Brynskov J, Thomsen OØ, et al. Individualised therapy is more cost-effective than dose intensification in patients with Crohn’s disease who lose response to anti-TNF treatment: a randomised, controlled trial. Gut 2014; 63: 919–927.
10. Wang SL, Ohrmund L, Hauenstein S, et al. Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum. J Immunol Methods 2012; 382: 177–188.
11. Lallemand C, Kavrochorianou N, Steenholdt C, et al. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists. J Immunol Methods 2011; 373: 229–239.
12. Perry M, Bewshea C, Brown R, et al. Infliximab and adalimumab are stable in whole blood clotted samples for seven days at room temperature. Ann Clin Biochem. 2015; doi: 10.1177/0004563215580001.
13. NICE. Crohn’s disease – Tests for therapeutic monitoring of TNF inhibitors (LISA-TRACKER ELISA kits, TNFa-Blocker ELISA kits, and Promonitor ELISA kits). Anticipated publication date: December 2015. (https://www.nice.org.uk/guidance/indevelopment/gid-dt24/consultation/crohns-disease-tests-for-therapeutic-monitoring-of-tnf-inhibitors-lisatracker-elisa-kits-tnfablocker-elisa-kits-and-promonitor-elisa-kits-consultation)
The authors
Mandy Perry*1 PhD, Tim McDonald1 FRCPath. PhD, Adrian Cudmore1, Tariq Ahmad2 MB ChB, DPhil, MRCP(UK)
1Department of Blood Sciences, Royal Devon and Exeter NHS Foundation Trust, Exeter, UK
2IBD Pharmacogenetics Research, University of Exeter, Exeter, UK
*Corresponding author
E-mail: mandy.perry@nhs.net
Anti-parietal cell antibodies
, /in Autoimmunity & Allergy, Featured Articles, Gastrointestinal Disorders /by 3wmediaby Dr Petraki Munujos The anti-parietal cell antibodies show one of the most distinctive fluorescent patterns in the autoantibody screening by indirect immunofluorescence. Although these antibodies react with a well known target antigen (H+/K+ ATPase) solely present in the parietal cells of the gastric gland, the use of combined tissue sections in the same reaction […]
Transforming molecular diagnostics workflows
, /in Featured Articles /by 3wmediaProfessor Jordi Vila, Head of Department of Clinical Microbiology, Hospital Clinic, School of Medicine, University of Barcelona, Spain, describes how a new, fully automated molecular diagnostic system, has the potential to improve productivity and turnaround times at his busy organ transplant reference laboratory in Barcelona, Spain.
The Hospital Clinic of Barcelona serves a local population of 540,000, in addition to being a National and International Centre of reference, providing the full range of medical and surgical specialties. The Hospital’s Department of Clinical Microbiology is also a reference laboratory for organ transplantation.
Operating 24 hours a day, seven days a week, the laboratory has experienced a growing workload in recent years, mainly associated with an increase in molecular biology assays, including viral loads for Human Immunodeficiency Virus type 1 (HIV-1), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) and Cytomegalovirus (CMV). In particular, the laboratory has observed an increase in HCV viral load requests, related to new treatment regimens, as well as an increase in CMV viral load requests for organ transplant patients.
The total number of viral load assays performed annually in the Barcelona laboratory for HIV-1, HCV, HBV and CMV are shown in figure 1.
The need for workflow improvements
Like many laboratories throughout Europe, the Virology Section at the Hospital Clinic of Barcelona must cope with this growing workload without any increase in staffing levels. As a result, there is a strong interest in workflow improvements as a means to increase productivity within the laboratory and to ensure the quality of generated results.
Speed and efficiency are particularly important when clinical decisions are dependent on the result, and an increase in automation, particularly in the disciplines of serology, molecular diagnostics and bacteriology, have played an important role in achieving greater speed and efficiency in the Clinical Microbiology Laboratory.
The aims of the laboratory’s investigations into increased automation and workflow improvements were to reduce turnaround times, to reduce waste (of time and reagents), to maximise the use of staff, space and equipment, to increase productivity and to reduce opportunities for error.
Limitations of current methods
When looking at potential areas for improvement, a number of drawbacks were observed in the current methods used for obtaining HIV-1, HCV, HBV and CMV viral loads. These methods require separate platforms for nucleic acid extraction, amplification and detection. Numerous steps are required to achieve the final result, which are quite labour intensive. In order to be cost effective, assays are performed in batches (table 1), which limits the number of assay runs performed in a week. This has a major impact on result turnaround times and has significant cost implications for urgent samples.
In addition to these limitations, all of the existing equipment and sample preparation is located in a small room where space is of a premium. As a result, working conditions are very crowded and some tasks, for example reagent preparation, need to be performed in an adjacent room, which is not ideal.
A new, fully automated system
An independent time/workflow analysis study was performed at the Hospital Clinic of Barcelona Virology Laboratory by Nexus Global Solutions (Plano, Texas, USA). This study compared workflows and time to results between current viral load methods and the new, fully automated DxN VERIS Molecular Diagnostics System (Beckman Coulter Inc.).
Launched at ECCMID 2015, the DxN VERIS Molecular Diagnostics System consolidates DNA extraction, amplification and detection on a single automated instrument. By reducing manual intervention and automating the process from sample loading to reporting of results, this system has the potential to transform virology laboratory workflows.
DxN VERIS assays are supplied in a unique, single cartridge system and all consumables and reagents are stored on-board the system, which reduces preparation time and effort. Unlike traditional plate-based systems, there is no need to batch assay runs and there are no empty wells, which reduces wastage and consumable costs. With true single sample random access, the DxN VERIS platform allows viral load assays to be performed as soon as they arrive in the laboratory and the short assay runtimes ensure rapid turnaround times.
Comparative performance studies at several DxN VERIS evaluation sites[1-13] have shown that the VERIS HBV, HCV, HIV-1 and CMV assays demonstrate comparable precision, sensitivity and linearity to a range of alternative, commercially available viral load methods.
Workflow study results
It was decided to run DxN VERIS samples as single sample random access, as intended by the manufacturers. This meant that samples could be loaded straight on to the DxN VERIS when they arrived in the laboratory, which is much faster than daily batch testing. The results of the comparative workflow study at the Hospital Clinic of Barcelona are shown in table 2 and figures 2 and 3.
In particular, the DxN VERIS workflow involved far fewer steps, especially pre-analytical steps, reduced hands-on time and fewer consumables. The time to the first result is greatly reduced compared to current methods and, notably, subsequent results are available every 2.5 minutes. For the current methods, results are not available until the end of the run.
During a normal working week, the DxN VERIS system allowed much faster turnaround of results, with all results being reported in under 24 hours (figure 3).
Workflow improvements
The DxN VERIS Molecular Diagnostics System offers some important workflow advantages compared to current methods for the determination of viral loads for HIV-1, HCV, HBV and CMV. For example, the DxN VERIS system allows continuous loading of samples, which eliminates the need for batching and, with true, single sample random access, it allows urgent samples to be added at any time. This is a particularly important aspect for us as a reference centre where urgent test requests can arrive at the laboratory at any time of day. The DxN VERIS system allows laboratories to perform assays for several viruses at the same time, on the same platform, which allows flexibility, and with adaptable racks, it also has the versatility to accept a variety of sample tube types.
As a fully automated system, the DxN VERIS system decreases the potential for human error and reduces turnaround times considerably compared to the current methods, which allows much faster reporting of results to service users. Unlike current methods, technicians are not required to pipette samples and reagents, which is an important ergonomic advantage. By reducing manual time requirements it will allow laboratories to achieve the most from existing staffing levels, helping to maximize productivity within the laboratory.
In addition to this, consolidation of extraction, amplification and detection for these four targets onto a single platform is an important consideration for laboratories, like this, where space is very limited.
The implementation of automated methodologies, such as this, has the potential to improve the quality and delivery of virology services and, for patients, it allows infectious disease results to be obtained at the earliest opportunity with high sensitivity and specificity.
For further information about the DxN VERIS Molecular Diagnostics System and the DxN VERIS assays currently available, please contact: Tiffany Page, Senior Pan European Marketing Manager Molecular Diagnostics, Email: info@beckmanmolecular.com or visit www.beckmancoulter.com/moleculardiagnostics.
References
1. Williams, JA, Rodriguez, J, Wang, Z et al (2014) Poster presentation, ESCV, Prague.
2. Drago, M, Franchetti, E, Fanti, D and Gesu, GP (2015) Poster presentation, EuroMedLab, Paris.
3. Zurita, S, Gutiérrez, F, Folgueira, MD et al (2015) Poster presentation, EuroMedLab, Paris.
4. Christenson, R, Maggert, K, Ruiz, RM et al (2015) Poster presentation, ECCMID, Copenhagen.
5. Trimoulet, P, Tauzin, B, Belloc, E et al (2015) Poster presentation, EuroMedLab, Paris.
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