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
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, /in Featured Articles /by 3wmediaMass control of soil-borne helminth infections: swings and roundabouts?
, /in Featured Articles /by 3wmediaIt is estimated that a quarter of the world’s population, predominantly those living in tropical and sub-tropical areas with inadequate sanitation, are infected by soil-borne nematode worms, the eggs of which are passed in human feces. The species of most concern are Ascaris lumbricoides, Trichuris trichiura and the hookworms Ancylostoma duodenale and Necator americanus. Their impact is insidious, with chronic infections resulting in increasingly debilitating micronutrient deficiencies that affect physical growth and mental development in children. Heavy hookworm infections are also associated with maternal morbidity and even mortality due to severe iron deficiency anemia.
The current WHO control strategy is first to examine fecal samples of older schoolchildren to establish the prevalence of infection in a community. If this exceeds 50%, all children from age one to fifteen, and ideally all women of child-bearing age except those in the first trimester of pregnancy, as well as workers in occupations with a high risk of infection, are treated with a benzimidazole antihelmintic twice a year. If the prevalence falls between 20% and 50%, treatment is annual. In areas with a prevalence lower than 20%, mass drug administration (MDA) is not recommended. However, several recently published articles have suggested that a more effective strategy would be community-wide MDA to eliminate helminth transmission entirely. Many such studies emphasize that, quite apart from the benefit for men and older women as well as those in communities with a lower prevalence, this approach would actually be a more efficient use of the limited resources available in the longer term. So what are the problems?
Firstly similar drugs have been used to control nematode worm infections in lifestock, and after several years of continual use high levels of resistance to the drugs developed; the same could occur in human populations. But there could be another problem. These antihelmintic drugs also kill ubiquitous and essentially innocuous parasites, and during the quarter of a century since Strachan first proposed his ‘Hygiene hypothesis’, suggesting that a lower exposure to microorganisms was linked to the noted rise in allergic conditions, there has been a plethora of publications supporting it. Many of these are based on very robust studies demonstrating and explaining the inverse relationship between parasitic infections and allergies and autoimmune disorders. Although it is admirable to alleviate the suffering caused by severe helminth infections, is it really prudent to eliminate all parasites and risk replacing the micronutrient deficiencies of less developed areas by the allergies and autoimmune diseases so common in the West?
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, /in Featured Articles /by 3wmediaEarly diagnosis of sepsis is essential for enabling appropriate treatment. PCT and MR-pro ADM have been shown to be independent biomarkers for sepsis and progression to septic shock, and simultaneous analysis seems to be more effective than the single marker approach.
by Dr S. Angeletti, M. De Cesaris, Dr A. Lo Presti, et al.
Introduction
Sepsis is a severe condition that represents the tenth most common cause of death in the USA. In Europe, sepsis occurs in more than 35% of the patients admitted in the intensive care unit. The mortality associated with sepsis is approximately 28% and it rises to 40–60% in cases of septic shock, despite adequate treatment administration. Nearly 9% of patients with sepsis experience severe sepsis and nearly 3% progress to septic shock leading to multi-organ failure. More than 50% of patients affected by septic shock do not survive [1–3]. Consequently, the rapid recognition and treatment of sepsis is mandatory to reduce both the mortality and the hospitalization with related costs [1-3].
Sepsis is commonly defined as the presence of infection in conjunction with the systemic inflammatory response syndrome (SIRS); severe sepsis, as sepsis complicated by organ dysfunction; and septic shock, as sepsis-induced acute circulatory failure characterized by persistent arterial hypotension despite adequate volume resuscitation and not explained by other causes [1, 4]. The diagnosis of sepsis and evaluation of its severity is complicated by the highly variable and non-specific nature of the signs and symptoms of sepsis [5]. However, the early diagnosis and stratification of the severity of sepsis is very important, increasing the possibility of starting timely and specific treatment [4, 6].
The gold standard for detection of bloodstream infections is blood culture. The time required for a positive blood culture result depends on the incubation time required for the culture to turn positive and the subsequent biochemical identification, along with an antibiotic sensitivity test, both of which usually take 48 h [7]. Furthermore, in some cases, blood culture results remain negative owing to empirical broad-spectrum antibiotics that are frequently started in the presence of SIRS and often continued for a prolonged time course despite the absence of clinical and microbiological data supporting a diagnosis of bacterial infection [4, 8]. Several studies have evaluated the diagnostic utility of various biomarkers, including ferritin, haptoglobin, interleukin 6, C-reactive protein (CRP) and procalcitonin (PCT) for suspected sepsis in the ICU patient population [9–11].
It remains difficult to differentiate sepsis from other non-infectious causes of SIRS [12] and there is a continuous search for better biomarkers of sepsis.
PCT is a polypeptide that has demonstrated the highest reliability in the early diagnosis of sepsis, severe sepsis or septic shock compared to other plasma biomarkers or clinical data alone [13]. Moreover, PCT has been advocated also to clarify the bacterial origin of some localized infections [14–15].
The mid-regional pro-adrenomedullin (MR-proADM) has been shown to play a decisive role in both the induction of hyper-dynamic circulation during the early stages of sepsis and the progression to septic shock [16–18], and recently it has been reported that MR-proADM differentiates sepsis from non-infectious SIRS with high specificity. Moreover, simultaneous evaluation of MR-proADM and PCT in septic patients increased the post-test diagnostic probabilities compared to the independent determination of individual markers [19–20]; probably the multimarker approach seems to be the more effective [14, 19].
The aim of the present study was to perform a focused evaluation of the role of the combination of PCT and MR-proADM in patients with severe sepsis and septic shock (SS) to differentiate it from patients with mild sepsis or SIRS for a prompt and specific treatment administration.
Methods
Patient and control characteristics
One hundred and seventeen patients with SS and 100 patients with SIRS, hospitalized at the University Hospital Campus Bio-Medico of Rome between the years 2012 and 2014, were enrolled in the study. The patients’ details are reported in Table 1.
Sepsis was defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference definition of sepsis [4] based on the presence of a recognized site of infection and evidence of a SIRS occurring when at least two of the following criteria are present: body temperature higher than 38°C or lower than 36°C, heart rate higher than 90 beats per minute, respiratory rate higher than 20 breaths per minute or hyperventilation as indicated by an arterial partial pressure of carbon dioxide (PaCO2) lower than 32 mm Hg and a white blood cell count of higher than 12,000 cells/mm3 or lower than 4,000.
Patients were classified according to clinical signs into SS and SIRS. Acute physiological and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores were computed. APACHE II scores in SS and SIRS patients were calculated by Medscape, APACHE II scoring system calculator [21]. The SOFA score was calculated only for SS patients to better define the severity of the sepsis [22–23]. The study was approved by the Ethics Committee of the University Hospital Campus Bio- Medico, Rome, Italy.
Blood culture
Blood samples for blood culture were collected when patients showed the symptoms and signs of SIRS [1, 2, 4]. Blood culture included three sets (time 0, time 30 and time 60 min) of one aerobic and one anaerobic broth bottles (Bactec Plus Aerobic/F, Bactec Plus Anaerobic/F, Beckton Dickinson) per patient drawn during 1-h period of clinically suspected bloodstream infection. Blood culture vials were incubated in the Bactec 9240 automated system (Beckton Dickinson). Blood culture samples that turned positive were immediately processed for Gram staining and cultivated. Bacterial identification was performed by MALDI-TOF, as previously described [24].
PCT and MR-proADM measurement
The plasma concentrations of PCT and MR-proADM were measured by an automated analyser using a time-resolved amplified emission method (Kryptor, Brahms AG), with commercially available assays (Brahms AG) [25].
Statistical analysis
Data was analysed using MedCalc 11.6.1.0 statistical package (MedCalc Software). Plasma levels of PCT and MR-proADM were log-transformed to achieve a normal distribution. The normal distribution of each marker concentration was tested by the Kolmogorov–Smirnov test. PCT and MR-proADM in patients with SIRS and SS were compared using the Mann–Whitney test. Multiple logistic regression analysis (stepwise method) using SS versus PCT and MR-proADM was performed and the odds ratio (OR) computed. For OR calculation variables were retained for P<0.05 and removed for P>0.1.
Receiver operating characteristic (ROC) analysis was performed among independent variables associated with SS to define the cut-off point for plasma PCT and MR-proADM and their diagnostic accuracy to predict SS [26]. Pre-test odds, post-test odds and the consequent post-test probability were computed to investigate whether the combination of PCT and MR-proADM improves post-test probability. Likelihood ratios were used as these tests are not prone to bias due to prevalence rates [27].
Results
Patients with SS and SIRS characteristics
The mean age of the 117 patients with SS (71 men and 46 women) was 69 ± 3 years (Table 1). The principal comorbidities of patients with SS and SIRS and the sources of bacteremia are summarized in Table 1. In patients with SS the average APACHE II score value was 19.8, corresponding to 24% risk of death and the average SOFA score was 6.8 corresponding to a predicted mortality of <33%. In patients with SIRS the APACHE II score was 7, corresponding to 6% risk of death (Table 1). SS was caused by Gram-negative pathogens in 63/117 (54%) of patients and in Gram-negative sepsis, E. coli (28/63; 44.4%) was the most frequent isolate. Gram-positive SS was present in 24/117 (20.5%) of cases and the most frequent pathogen was S. aureus (14/24; 58.3%), whereas C. albicans was the most frequent isolate in yeast-positive cultures (10/117; 8.5%) and blood cultures were polymicrobial in 20/117 (17%) cases. Bacterial isolates from positive blood culture are reported in Table 2.
PCT and MR-proADM in patients with SS and SIRS
Median values, interquartile ranges (25th percentile and 75th percentile) and Mann–Whitney comparison of PCT and MR-proADM analysed in patients with SS and SIRS are reported in Table 3. PCT and MR-proADM values were significantly higher in patients with SS than SIRS (P<0.0001) (Table 3 and Figure 1). ROC curve and AUC analysis of PCT and MR-proADM in patients with SS
In SS patients, the area under curve (AUC) values of PCT and MR-proADM are reported in Table 4. Based upon ROC curve analysis and AUC characteristics, PCT and MR-proADM were considered applicable for sepsis diagnosis at the cut-off values of 0.5 ng/mL and 1 nmol/L, respectively (Table 4 and Figure 2).
Multiple logistic regression analysis
Multiple logistic regression analysis using SS as the dependent variable and PCT and MR-proADM as independent variables is reported in Table 5. Patients with MR-proADM >1 nmol/L have ~195 times the probability of being affected by SS than patients with SIRS, and patients with PCT values >0.5 ng/mL have the probability of developing SS 49 times more than SIRS.
Combined PCT and MR-proADM measurement in SS diagnosis: post-test probability calculation
In patients with SS, PCT and MR-proADM used as single markers have a post-test probability of 0.964 and 0.936, respectively. The combination of PCT and MR-proADM resulted in a higher value of post-test probability, 0.996 (Table 4).
Discussion
The early diagnosis and stratification of the severity of sepsis are essential, increasing the possibility of starting timely the specific treatment, especially in patients affected by SS. In this study, the combined measurement of PCT and MR-proADM in patients with SS was evaluated in order to establish the advantage derived from the use of a multimarker rather than a single marker approach.
PCT has been described as a reliable marker in the early diagnosis of sepsis compared to other plasma biomarkers or clinical data alone [13, 14, 19]. MR-proADM has been used as marker of disease severity in different clinical setting and recently its combination with PCT in bacterial infections and sepsis has been evaluated [28–32, 14, 19]. The combination of PCT and MR-proADM could allow the simultaneous evaluation of the presence of a bacterial infection as well as of the severity of this infection, giving to the ward clinicians a first useful indication waiting for blood culture positivity.
Results from this study demonstrated that in patients with SS, PCT and MR-proADM values are significantly higher than patients with SIRS. ROC curve analysis of PCT and MR-proADM demonstrated a high diagnostic accuracy of these two markers in SS diagnosis at the cut-off value of 0.5 ng/mL and 1 nmol/L, respectively. The logistic regression analysis showed higher OR values for both markers indicating a significant increased risk of having SS when these markers are higher than the cut-off values established. Furthermore, the combination of the two markers leads to a very high post-test probability value of about 99.6%.
These data confirmed the important role of the combination of PCT and MR-proADM in the diagnosis and prognosis of patients with sepsis rather than the single marker approach, because it combines the diagnostic ability of PCT with the prognostic value of MR-proADM, as already described in localized bacterial infections and not complicated sepsis [14, 19].
In conclusion, this study further support the advantage derived from the multi-marker approach in sepsis diagnosis and prognosis, especially in critically ill patients.
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12. Vänskä M, Koivula I, Jantunen E, Hämäläinen S, Purhonen AK, et al. IL-10 combined with procalcitonin improves early prediction of complications of febrile neutropenia in hematological patients. Cytokine 2012; 60: 787–792.
13. Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C. High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 1993; 341: 515–518.
14. Angeletti S, Spoto S, Fogolari M, Cortigiani M, Fioravanti M, et al. Diagnostic and prognostic role of procalcitonin (PCT) and MR-pro-Adrenomedullin (MR-proADM) in bacterial infections. APMIS 2015; 123: 740–748.
15. Kojic D, Siegler BH, Uhle F, Lichtenstern C1, Nawroth PP, et al. Are there new approaches for diagnosis, therapy guidance and outcome prediction of sepsis? World J Exp Med. 2015; 5: 50–63.
16. Hirata Y, Mitaka C, Sato K, Nagura T, Tsunoda Y, et al. Increased circulating adrenomedullin, a novel vasodilatory peptide, in sepsis. J Clin Endocrinol Metab. 1996; 81: 1449–1153.
17. Kitamura K, Kangawa K, Kawamoto M, Ichiki Y, Nakamura S, et al. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem Biophys Res Commun. 2012; 425: 548–555.
18. Zhou M, Ba ZF, Chaudry IH, Wang P. Adrenomedullin binding protein-1 modulates vascular responsiveness to adrenomedullin in late sepsis. Am J Physiol Regul Integr Comp Physiol. 2002; 283: R553–560.
19. Angeletti S, Battistoni F, Fioravanti M, Bernardini S, Dicuonzo G. Procalcitonin and mid-regional pro-adrenomedullin test combination in sepsis diagnosis. Clin Chem Lab Med. 2013; 51: 1059–1067.
20. Suberviola B, Castellanos-Ortega A, Ruiz Ruiz A, Lopez-Hoyos M, Santibañez M. Hospital mortality prognostication in sepsis using the new biomarkers suPAR and proADM in a single determination on ICU admission. Intensive Care Med. 2013; 39: 1945–1952.
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The authors
S. Angeletti*1 MD, M. De Cesaris1, A. Lo Presti2 PhD, M. Fioravanti1, F. Antonelli1, R. Ottaviani1, L. Pedicino1, A. Conti1, A. M. Lanotte1, M. Fogolari1 MD, M. Ciccozzi2 PhD, G. Dicuonzo1 MD
1Clinical Pathology and Microbiology Laboratory, University Hospital Campus Bio-Medico of Rome, Italy
2Department of Infectious, Parasitic, and Immune-Mediated Diseases, Epidemiology Unit, Reference Centre on Phylogeny, Molecular Epidemiology, and Microbial Evolution (FEMEM), National Institute of Health, Rome, Italy
*Corresponding author
E-mail: s.angeletti@unicampus.it
Sepsis: earlier organism identification using MALDI-TOF
, /in Featured Articles /by 3wmediaSepsis is a life threatening inflammatory disorder and the immune systems response to infection. It is one of the leading causes of death in hospitalized patients worldwide with 1.8 million cases annually. Improvement in survival remains contingent on early recognition of the causative organism to enable targeted antimicrobial therapy.
by Kelly Marie Ward and Rhian Harris
Sepsis incidence
Sepsis is a life threatening inflammatory disorder and the immune systems response to infection [1]. It is one of the leading causes of death in hospitalized patients worldwide with 1.8 million cases annually [2]. Each year, 37 000 deaths are caused by sepsis in the UK [2–4]. Mortality rates remain between 25–30% for severe sepsis and 40–70% for septic shock, despite advances in pharmacotherapy and supportive care [1] and various campaigns, e.g. the Surviving Sepsis Campaign (SSC) [3]. This is mainly due to poor identification and delayed interventions [3]. Data from the SSC showed a mortality rate of 39.8% among 15 022 patients and 39.8% of those admitted to critical care in England and Wales die in hospital [2]. A hospital admission with severe sepsis places the patient at a level of risk 6–10-fold greater than admission with an acute myocardial infarction and 4–5 times greater than if they had suffered an acute stroke [2].
What is sepsis?
The American College of Chest Physicians and the Society of Critical Care Medicine classified the continuum of an inflammatory response to microorganisms as ‘systemic inflammatory response syndrome’ (SIRS) [1]. SIRS is a collection of signs that show the body is reacting to a range of injuries or illnesses and it is not specific to infection [3]. It is identified when two of the following symptoms – fever, tachycardia, tachypnea and leukopenia are met in the absence of an infection [3].
Uncomplicated sepsis is the presence of an infection in association with SIRS [1] in the absence of organ dysfunction [4]. Bacteria that cause infection can enter the body via breaks in the skin, catheters and underlying infections in the urinary, respiratory or gastrointestinal tract [5]. Sepsis can be defined as ‘a systemic disease that is caused by the spread of microorganisms and their toxins via the circulating blood’ [6]. The endo and exotoxins produced by different organisms often lead to an inflammatory response of varying severities [7]. Severe sepsis occurs when SIRS is accompanied by infection and organ dysfunction [4]. Figure 1 taken from Royal College of Physicians: Acute Care Toolkit 9: Sepsis; September 2014 [4] demonstrates this balance.
Pathophysiology of sepsis
The pathophysiology of sepsis involves a complex interaction of proinflammatory and anti-inflammatory mediators in response to pathogen invasion [1]. When an infectious agent invades the host, an innate response is triggered via toll-like receptors (TLR) [8]. These are trans-membrane proteins with the ability to promote signalling pathways downstream and trigger cytokine release, neutrophil activation and stimulation of endothelial cells [8]. The cytokines such as interleukin (IL)-1 and IL-6 are released from the cells where inflammatory reactions have commenced. They stimulate lymphocytes and mononuclear cells to produce further cytokines, resulting in the recruitment and migration of further cells to the site or organ where inflammation is occurring [9]. This leads to endothelium damage, vascular permeability, microvascular dysfunction, coagulation pathway activation and impaired tissue oxygenation resulting in the cascade of sepsis [1]. There is activation of humoural and cell-mediated immunity with specific B and T cell responses and both pro and anti-inflammatory cytokine release [8]. Adaptive immunity is triggered and the inflammatory cascade of sepsis occurs where the balance is shifted towards cell death and a state of relative immunosuppression and end organ dysfunction ensues with hemodynamic changes causing elevated cardiac output and generalized vasodilation described as shock [8]. As the inflammatory response progresses, myocardial depression is more pronounced resulting in a falling cardiac output. There is capillary leak and pulmonary edema that may progress to acute lung injury. Renal failure then follows accompanied by alterations in the coagulation cascade towards a pro-coagulant and antifibrinolytic state. The development of ‘disseminated intravascular coagulation’ (DIC) in severe sepsis is a predictor of death and the development of multiorgan failure [8]. The respiratory, genitourinary and gastrointestinal systems are most commonly infected and pneumonia is the most common presentation leading to sepsis [1].
Clinical presentation and diagnosis
There are a variety of symptoms that can indicate sepsis, including fever, chills, decreased blood pressure, shaking, skin rash, confusion and a rapid heartbeat [10].The clinical diagnosis of sepsis is most often made before culture results are available and although localized signs and symptoms may be present, organ hypoperfusion or shock can occur without the knowledge of the cause [1]. Fever is the most common manifestation of sepsis and 40% of those patients will have hypotension [1].
A vast array of laboratory tests are required for the diagnosis and management of sepsis including full blood counts, basic metabolic panels, lactate and liver enzyme levels and C-reactive protein. In the Microbiology laboratory we would expect to receive, blood cultures: two peripheral and from each indwelling catheter, urine, stools if symptoms of diarrhoea, sputum and skin and soft tissue for culture if clinically significant [1] Currently blood cultures are the definitive diagnosis tool when septicemia is suspected [11]. Blood culture systems have evolved over time to ensure optimum isolation of any organisms present by adding different nutrients, introduction of automated systems and increasing the detection rate of a positive as a result of new software [12]. They are used to detect the presence of any microorganisms present by providing optimum conditions for growth. However in 50–65% of patients the blood culture is often negative [1].
Early management and identification of infectious cause
Due to the high mortality rates the early identification and management of sepsis is crucial and requires respiratory stabilization followed by fluid resuscitation, vasopressor therapy, infection identification and control and prompt antibiotic administration [1].
The SSC published ‘The Resuscitation Bundle’ which comprised a set of tasks to be to be completed within the first 6 hours after the clinical identification of sepsis [2]. The first four tasks were:
These tasks involve the Pathology laboratory and it was established that systems within healthcare environments needed to be well designed and implemented to ensure that the appropriate investigations, equipment and treatments were available at the point of care [2].
The length of time that it takes for the correct identification of the causative organism has many ramifications both clinically and financially. Empiric antibiotic therapy is based on the most likely source, clinical context, recent antibiotic use and local resistance patterns. This should be narrowed when the causative agent has been identified to reduce the risk of resistance or superinfection [1]. The length of time the patient is prescribed such antibiotics may be reduced if the causative organism is characterized sooner, and inappropriate therapy changed accordingly. The use of targeted narrow spectrum antibiotics might reduce bed days resulting in a financial saving. Using antibiotics more than is required in both humans and animals has resulted in the increased emergence of antibiotic resistance [13]. New antibiotics are produced very slowly and as a result it is important to limit any likelihood of the spread of resistance between organisms by only prescribing the antibiotic necessary [14]. Also early appropriate antibiotic therapy is associated with improved clinical outcomes [1]. The antibiotics should be administered within 1 hour of suspected sepsis. In septic shock early antibiotic therapy increases survival and for each hour this is delayed the survival rates decrease by 8% [1]. The possible benefits to the patient in terms of improved treatment of infections, the potential to reduce costs and an attempt to reduce the emergence of antibiotic resistance have led to the development and research into more rapid diagnosis techniques.
MALDI-TOF and the Bruker Sepsityper blood culture kit
Matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) spectroscopy can identify organisms from intact cells based on the profile of different proteins and relative molecular mass [15]. A smear of the cultured organism is placed onto a stainless steel target plate, with matrix placed over the top. Matrix is used as it prevents fragmentation of higher mass molecules. The laser is fired at the smear generating a cloud of ions which are accelerated up the flight tube to the detector, where the time of flight is converted to Daltons (Da)/molecular mass. The heavier the molecular mass of the ion the greater the time of flight (Fig. 2).
This is also known as proteomic profiling. A spectrum is produced for each organism based on their mass/charge ratio, which is determined by the different molecular mass and charge of the ions present for the organism in question [15]. A spectrum is produced with a variety of peaks each one representing a different molecular fragment which has been released as a result of the laser desorption [15]. This spectrum is then compared to the database for possible matches and is scored based on the number of peaks that match the corresponding organism (Fig. 3).
This method can be used to identify bacteria, yeasts, moulds, mycobacteria and Nocardia to species level using species-specific spectral patterns [16]. Using this method, identification from a bacterial culture can be achieved in 30–60 seconds.
MALDI-TOF spectroscopy can also be used to identify organisms directly from blood culture bottles using the Sepsityper kit extraction method (Fig. 4). This takes approximately 30 minutes and allows accurate identification to species level on day 1 of the bottle being flagged as positive. The use of the Sepsityper kit could enhance task three of the SSC Resuscitation bundle by allowing earlier targeted therapy. Components within the blood culture such as red cells, white blood cells and serum can interfere with the analysis resulting in the formation of additional spectral peaks [17]. These peaks will not be found in the database and will result in difficulty in interpreting the results, which is why the extraction kit by Bruker has been developed. The development of this kit has allowed purification and extraction to be carried out to optimize recovery of the bacteria present in the blood culture sooner. This is carried out by a series of centrifugation steps to separate any organisms present from the blood and fluid present in the blood culture and also formic acid to breakdown the cell wall of the organism to aid identification using MALDI-TOF technology (Bruker – Introduction for use Maldi Sepsityper kit (Accessed 2015).
Our laboratory evaluated the use of the Sepsityper kit to identify the causative organism direct from the positive blood culture bottle using MALDI-TOF spectroscopy. The results were retrospectively analysed to determine if there would have been a change to the antibiotic therapy if this method was in routine use.
Study results
Two hundred and thirty-six positive blood cultures were analysed retrospectively and compared against current laboratory methods. The results are shown in Table 1.
Table 1 shows the percentage of successful identifications achieved by using the Bruker Sepsityper method. The percentage of blood cultures that achieved successful identification within 1 hour of becoming positive was 75.42% (green plus yellow rows). A score of above 1.8 indicates a secure genus and probable species identification (green row) [18], a score between 1.6 and 1.8 indicates probable species identification (yellow row) [18]. Any score below 1.6 cannot be accepted as a reliable identification (red row). There was a 93.33% agreement of identification between the Bruker Sepsityper kit or direct MALDI-TOF identification versus the BD Phoenix and other conventional laboratory methods.
The previous antibiotic treatment, the clinical history of the patient and the identification of the organism produced by the Bruker Sepsityper kit on day one was analysed retrospectively by the consultant microbiologist to determine if there would have been any clinical impact if the identification of the organism had been known on day 1. As the organism that is causing the infection is not known immediately, patients are started on broad-spectrum or a combination of antibiotics when bacteremia is suspected; however, incorrect or insufficient therapy has been associated with increased mortality, morbidity and increased hospital stay [19]. The consultant microbiologist determined that 26 (11%) out of the 236 blood cultures analysed would have indicated a requirement for the patient to have their antibiotic therapy altered in some way. Sixteen of the 26 positive blood cultures indicated that the patients’ antibiotic therapy could be reduced from a broad-spectrum antibiotic to a narrower spectrum antibiotic. This can have huge cost savings implications as well as reduce the likelihood of resistance emerging against broad-spectrum antibiotics [20]. Knowing the identification of the organism on the day the blood culture bottle is flagged as positive enables the antimicrobial therapy to be changed accordingly therefore helping to reduce the emergence of resistance, provide targeted therapy for better treatment outcomes and reduce bed days spent in hospital. Improvement in survival remains contingent on the early recognition and management of severe sepsis and septic shock [1].
References
1. Gauer RL. Early Recognition and Management of Sepsis in Adults: The First Six Hours. Am Fam Physician. 2013; 88: 44–53.
2. Daniels R. Surviving the First Hours In Sepsis: getting the basics right (an intensivists perspective). J Antimicrob Chemother. 2011; 66(Suppl 2): ii11–23.
3. McClelland H, Moxon A. Early identification and treatment of sepsis. Nursing Times 2014; 110: 14–17. (http://www.nursingtimes.net/Journals/2014/01/17/q/v/z/220114-Early-identification-and-treatment-of-sepsis.pdf)
4. Royal College of Physicians. Acute Care Toolkit 9: Sepsis; September 2014. (https://www.rcplondon.ac.uk/sites/default/files/acute_care_toolkit_9_sepsis.pdf)
5. Public Health England. Investigation of blood cultures. Bacteriology 2014; B37(8). (https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/372070/B_37i8.pdf)
6. Odeh M. Sepsis, septicaemia, sepsis syndrome, and septic shock: the correct definition and use. Postgrad Med J. 1996; 72(844): 66.
7. Martin GS. Sepsis, severe sepsis and septic shock: changes in incidence, pathogens and outcomes. Expert Rev Anti-infect Ther. 2012; 10(6): 701–706.
8. Ventetuolo CE, Levy MM. Sepsis: a clinical update. Clin J Am Soc Nephrol. 2008; 3: 571–577.
9. Roitt I, Brostoff J, Male D. Cell migration and inflammation. In: Cook L, Immunology, 4th ed. Mosby 1998.
10. Severe sepsis/septic shock, recognition and treatment protocols. Stony Brock Medicine 2013. (http://www.survivingsepsis.org/sitecollectiondocuments/protocols-sepsis-treatment-stony-brook.pdf)
11. Previsdomini M, Gini M, et al. Predictors of positive blood cultures in critically ill patients: a retrospective evaluation. Croat Med J. 2012; 53(1): 30–39.
12. Zadroga R, Williams DN, et al. Comparison of 2 blood culture media shows significant differences in bacterial recovery for patients on antimicrobial therapy. Clin Infect Dis. 2012; 56(6): 790–797.
13. Rao GG. Risk factors for the spread of antibiotic-resistant bacteria. Drugs 1998; 55(3): 323–330.
14. Guidos RJ. Combating antimicrobial resistance: policy recommendations to save lives. Clin Infect Dis. 2011; 52(5): 397–428.
15. Carbonnelle E, Beretti JL, et al. (2007). Rapid identification of Staphylococci isolated in clinical microbiology laboratories by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2007; 45(7): 2156–2161.
16. Stevenson LG, Drake SK, Murray PR. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol. 2010; 48(2): 444–447.
17. Lagacé-Wiens PRS, Adam HJ, et al. Identification of blood culture isolates directly from positive blood cultures by use of matrix-assisted laser desorption ionization–time of flight mass spectrometry and a commercial extraction system. J Clin Microbiol. 2012; 50(10), 3324–3328.
18. El-Bouri K, Johnston S, et al. Comparison of bacterial identification by MALDI-TOF mass spectrometry and conventional diagnostic microbiology methods: agreement, speed and cost implications. Br J Biomed Sci 2012; 69(2): 47–55.
19. Kollef MH. Broad-spectrum antimicrobials and the treatment of serious bacterial infections: getting it right up front. Clin Infect Dis. 2008; 47(1): 3–13.
20. Rüttimann S, Keck B, et al. Long-term antibiotic cost savings from a comprehensive intervention program in a medical department of a university-affiliated teaching hospital. Clin Infect Dis. 2004; 38(3): 348–356.
The authors
Kelly Marie Ward* MSc, FIBMS; Rhian Harris MSc, AIBMS
Royal Glamorgan Hospital Microbiology Laboratory, Cwm Taf University Health Board, Llantrisant, Glamorgan, UK
*Corresponding author
E-mail: Kelly.Ward@wales.nhs.uk
Molecular diagnostics – ushering new frontiers in allergy immunotherapy
, /in Featured Articles /by 3wmediaMolecular allergy (MA) diagnostics determines the sensitivity of allergy patients at a molecular level. This is achieved by using recombinant allergenic molecules to determine allergic response, as opposed to the traditional method of testing crude extracts of potential allergenic sources. Although MA diagnostics remains an emergent technique, it promises to revolutionize the diagnosis and treatment of allergies.
Classes of allergy
An allergy “is an overreaction by the human immune system to certain substances in the environment that are usually harmless.” Allergic diseases are categorized into four main types, based on reaction mechanism and time – from contact with an allergen until the appearance of the first symptoms. Clinical manifestations of allergy range from mild irritation through to potentially fatal anaphylactic shock.
The most common allergies are Type I , which involve an immediate reaction. Examples of Type I allergies include hay fever, allergies to animal hair, insect venom, latex, dust mites, asthma and hives. Allergic reactions to medication such as local anesthetics and antibiotics are also considered Type I, as are food allergies.
Other allergy types are both rare and take longer before symptoms appear: Type II (cytotoxic, such as blood transfusion reactions), Type III (immune complex allergies like arthritis and nephritis) and Type IV (delayed-onset allergies with cellular immune reactions such as organ transplant rejection).
A growing and costly problem
Allergic diseases affect up to 25% of the population in industrialized countries and their incidence is rising, especially in children. In the US, allergic diseases comprise the fifth leading chronic disease among all ages, and the third most common chronic disease in children under 18 years. Food allergies pose their own specific challenges. In the US, between 1997 and 2007, “the prevalence of reported food allergy increased 18% among children.” In Europe, more than 17 million people have a food allergy, and hospital admissions for severe reactions in children have risen seven-fold over the past decade, according to the European Academy of Allergy and Clinical Immunology (EAACI).
The economic costs of allergies include medical bills, lost work and missed school as well as what is often a dramatic reduction in the quality of life. The cost of food allergies alone in the US is $25 billion a year. In Europe, research indicates that avoidable indirect costs per patient insufficiently treated for allergy are 2,405 euros per year due to absence from work and reduced working capacity.
IgE antibody, a 1960s biomarker
The discovery of the immunoglobulin (IgE) antibody in the 1960s was a revolution in its time, as it provided a specific biomarker to identify allergies triggered by allergens. Traditional IgE antibody tests such as skin prick tests (SPT) or in vitro specific IgE (sIgE) tests depend on extracts of allergenic and non-allergenic molecules from an allergenic source. Even now, most patients are diagnosed by such methods. However, they are time consuming and imprecise, especially for patients with complex presentations such as multiple sensitization.
Cross-reactivity, other challenges
Allergen components are classified by protein families based on function and structure and allergic reactions are caused by response to individual proteins which make up the allergen source. The extent of reaction varies from one protein to another, as well as between different subjects.
Another key problem with traditional tests involves the stability of an allergen. Allergens which are stable to heat and digestion are associated with severe clinical reactions, whereas heat and digestion labile molecules are likelier to cause milder, local reactions or even be tolerated.
For allergy patients, cross-reactive components (where proteins share similar structures) provoke unpleasant and sometimes severe symptoms. However, sensitization to a cross-reactive component does not indicate a primary cause. It is the latter which must be investigated thoroughly and identified in order to diagnose and manage an allergy.
MA diagnostics: precision targeting
MA diagnostics is now offering answers to such quandaries. Rather than testing for reaction to sources, MA diagnostics tests directly for sensitivity to specific proteins – namely the allergen components. In other words, one of the most important clinical assets of MA diagnostics is its ability to reveal whether the sensitization is genuine in nature (primary, species-specific) or if it is due to cross-reactivity to proteins with similar protein structures. This, in turn, may help to evaluate the risk of reaction on exposure to different allergen sources.
For clinicians, component testing enables identification of a genuine allergy as opposed to symptoms provoked by cross-reactivity (i.e. reactions due to similar protein structures). This allows them to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thereby improve the management of an allergy.
As a result, MA diagnostics is the best way to achieve precision in searching for the primary allergen component. It also enables the design of an accurate and effective component-resolved sensitization profile for each allergy patient. Apart from resolving genuine versus cross-reactive sensitization, MA diagnostics can in certain cases also assess the risk of severe, systemic versus mild, local reactions.
Recombinant technology and the fight against allergy
MA diagnostics was made possible by the growth of DNA technology in the late 1980s. By 1991, scientists were reporting that recombinant allergens proved useful for the “setup of diagnostic tests that allow the discrimination of different IgE-binding patterns.”
Recombinant technology allows “full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency” of allergens. These parameters had not been possible to assay and standardize for extract-based products. Finally, recombinant technology also permits bulk production of wild type molecules for diagnostics.
Over the 1990s and 2000s, DNA sequences of most common allergens were isolated and produced as recombinant molecules. By 2013, a total of “more than 130 allergenic molecules” were commercially available” for in vitro testing.
Due to the rapid growth in the number of allergens identified, a systematic allergen nomenclature, approved by the World Health Organization (WHO) and International Union of Immunological Species (IUIS) has been established. The so-called Allergen Nomenclature Subcommittee is in charge of developing and maintaining the nomenclature for allergenic molecules, as well as a comprehensive database of known allergenic proteins (available at www.allergen.org).
Singleplex and multiplex platforms
The process of diagnostic testing is relatively straightforward. The presence of IgE antibodies against allergenic molecules is determined using two kinds of measurement platforms. The singleplex platform consists of one assay per sample and allows a clinician to select allergenic molecules deemed necessary for diagnosis – as determined by the clinical history of the patient. The multiplex approach, which consists of multiple assays per sample, allows characterization of the IgE response against a broad array of pre-selected allergens on a chip independently of the clinical history.
Microarray-based testing
The near-term future promises a rapid influx of new data given growth in the availability of microarray-based tests. This will allow the design of stronger and larger number of studies “to critically evaluate their diagnostic and prognostic power over existing test modalities.”
A key advantage of microarray-based testing is that it requires only small volumes of serum samples to determine specific-IgE antibodies against multiple recombinants. The technique has also proven its credibility. In August 2010, the journal ‘Clinical and Experimental Allergy’ observed that the “performance characteristics of allergens so far tested are comparable with current diagnostic tests.”
The availability of recombinant allergens and the development of protein microarray-based immunoassays developed side-by-side over the 2000s and have now begun to cross-fertilize one another. In 2011, the ‘Journal of Allergy and Clinical Immunology’, the official publication of the American Academy of Allergy, Asthma & Immunology, noted that the “long-anticipated wider application” of recombinant allergens and protein microarray-based immunoassays to allergy diagnosis “has recently begun to accelerate,” with a demonstration of the potential “for greater resolution between clinical reactivity and asymptomatic sensitization.”
Printed microarrays: a promising new frontier
One area of growing interest is the use of printed microarrays as a platform for cellular assays. For example, protein microarray (PM) appears to be a powerful alternative to costly or labour-intensive diagnostics for large-scale detection of allergen-specific IgE. A recent study established a proof-of-concept to demonstrate that “coupling the diversity of protein array with the biological output of basophilic cells is a feasible proposition,” and avoids “costly, cumbersome and time-consuming” procedures for purification.
MA diagnostics and personalized medicine
With special attention paid to species-specific or primary sensitization and cross-reactivity, MA diagnostics is also becoming a tool to determine the right treatment for a patient at the right time – in other words, a frontier for personalized medicine. Data from MA diagnostics paves the way to individualize treatment actions, including advice on targeted allergen exposure reduction and specific immunotherapy (SIT). Nevertheless, clinicians recommend that in vitro tests should be evaluated together with clinical history, because allergen sensitization does not necessarily imply clinical responsiveness.
Allergen-specific immunotherapy (SIT) is the only antigen-specific and disease-modifying approach for the treatment of allergy. Though the symptoms of allergy can often be effectively suppressed using various drugs, it has been known since the late 1990s that “only allergen immunotherapy is able to impact on the underlying immune mechanism and leads to long-lasting change in the course of allergic disease.” It is based on the therapeutic administration of the disease-causing allergens to allergic patients.
In the past, several disadvantages limited the applicability of SIT, among them unwanted effects, poor efficacy and specificity as well as inconvenient application. Most of these were related to the poor quality of natural allergen extracts.
Due to recent progress in molecular allergen characterization, “new allergy vaccines based on recombinant allergens, recombinant hypoallergenic allergen derivatives and allergen-derived T cell peptides have entered clinical testing and hold promise to reduce the side-effects and to increase the specificity as well as the efficacy of SIT.”
Towards refined immunotherapy
Today, the focus of attention is on what has become known as ‘refined immunotherapy’, based on the use of peptides derived from allergen surfaces that exhibit reduced, allergen-specific IgE as well as T cell reactivity. When fused to non-allergenic carriers, these peptides provide allergen-specific protective IgG responses with T cell help from a non-allergenic carrier molecule. Recent data shows that such peptide vaccines “can bypass allergen-specific IgE as well as T cell activation and may be administered at high doses without IgE- and T cell-mediated side-effects.”
Such peptide vaccines are being evaluated in clinical trials. If successful, it may well be possible to develop safe forms of SIT as effective alternatives to drug-based allergy treatment.
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
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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