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.
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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.
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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
Combined PCT and MR-proADM testing in the diagnosis and prognosis of severe sepsis and septic shock
, /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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22. Vincent JL, de Mendonça A, Cantraine F, Moreno R, Takala J. Use of the SOFA score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. Working group on “sepsis-related problems” of the European Society of Intensive Care Medicine. Crit Care Med. 1998; 26: 1793–1800.
23. Kajdacsy-Balla Amaral AC, Andrade FM, Moreno R, Artigas A, Cantraine F, Vincent JL. Use of the sequential organ failure assessment score as a severity score. Intensive Care Med. 2005; 31: 243–249.
24. Angeletti S, Dicuonzo G, D’Agostino A, Avola A, Crea F. Turnaround time of positive blood cultures after the introduction of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. New Microbiol. 2015; 38: 379–386.
25. Christ-Crain M, Morgenthaler NG, Struck J, Harbarth S, Bergmann A, Müller B. Mid-regional pro-adrenomedullin as a prognostic marker in sepsis: an observational study. Crit Care 2005; 9: R816–824.
26. Florkowski CM. Sensitivity, specificity, receiver-operating characteristic (ROC) curves and likelihood ratios: communicating the performance of diagnostic tests. Clin Biochem Rev. 2008;29(Suppl 1): S83–7.
27. Albert A. On the use and computation of Likelihood ratios in clinical chemistry. Clin Chem. 1982; 28: 1113–1119.
28. Maisel A, Mueller C, Nowak R, Peacock WF, Landsberg JW, et al. Mid-Region pro-hormone markers for diagnosis and prognosis in acute dyspnea. J Am Coll Cardiol. 2010; 55: 2062–2076.
29. Maisel A, Mueller C, Nowak R, Peacock WF, Ponikowski P, et al. MidRegion prohormone adrenomedullin and prognosis in patients presenting with acute dyspnea. J Am Coll Cardiol. 2011; 58: 10572–10567.
30. Paecock WF, Nowak R, Christenson R, DiSomma S, Neath SX, et al. Short-term mortality risk in emergency department acute heart failure. Acad Emerg Med. 2011; 18: 947–958.
31. Travaglino F, De Berardinis B, Magrini L, Bongiovanni C, Candelli M, et al. Utility of procalictonin (PCT) and mid regional pro-adrenomedullin (MR-proADM) in risk stratification of critically ill febrile patients in emergency department (ED). A comparison with APACHE II score. BMC Infect Dis. 2012; 12: 184.
32. Hagag AA, Elmahdy HS, Ezzat AA. Prognostic value of plasma pro-adrenomedullin and antithrombin levels in neonatal sepsis. Indian Pediatr. 2011; 48: 471–473.
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
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.
6. Gilfillan, R, Wang, Z, Xu, Y et al (2014) Poster presentation, ECCMID, Barcelona.
7. Xu, Y, Gilfillan, R, Wang, Z et al (2014) Poster presentation, ESCV, Prague.
8. Mengelle, C, Sauné, K, Haslé, C et al (2014) Poster presentation, RICAI.
9. Mengelle, C, Sauné, K, Haslé, C et al (2015) Poster presentation, ECCMID, Copenhagen.
10. Silvestro, A, Duan, H, Lim, S et al (2014) Poster presentation, ECCMID, Barcelona.
11. Li, Q, Williams, J, Maggert, K et al (2014) Poster presentation, ECCMID, Barcelona.
12. Xu, Y, Dineen, S, Annese, V et al (2014) Poster presentation, ESCV, Prague.
13. Williams, JA, Rodriguez, J, Wang, Z et al (2014) Poster presentation, ECCMID, Barcelona.
Simplexa HSV 1& 2 Direct
, /in Featured Articles /by 3wmedia