JOURNAL TRANSCRIPT
Biomarkers of fever: f rom bench to bedside m. limper
Biomarkers of fever: from bench to bedside M. Limper Rotterdam, 2014
Financial support for the publication of this thesis was kindly provided by: Bristol-Myers Squibb, ViiV Healthcare, Gilead, AbbVie, Janssen, ThermoFisher Scientific. ISBN: 978-94-6169-476-8 Cover design by: Optima Print Rotterdam Design and lay-out: Optima Print Rotterdam No part of this thesis may be reproduced or transmitted in any form by any means without prior permission of the author, or when appropriate, of the scientific journal in which parts of this thesis have been published. © 2014 M. Limper Cover: “Quarantainehuis” in Curaçao. In 1874, after the revision of the Quarantine Law, this institution arranged for isolation of sailors suffering from yellow fever. Suspected ships were recognizable by their so-called “Yellow Jack”, a yellow flag, that was required in case of suspected yellow fever cases. Yellow fever was considered one of the most contagious diseases of that time. In the “Quarantainehuis”, suspected patients were incorporated for observation. In 1917, 17 years after the discovery of a vaccine against yellow fever, the building no longer had a quarantine function and was abandoned ever since.
Biomarkers of fever: from bench to bedside Biomarkers van koorts: van lab naar kliniek Proefschrift ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. dr. H.A.P. Pols en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op 28 maart 2014 om 9.30u te Woudestein door Maarten Limper geboren te Amsterdam
Promotiecommissie Promotores:
Prof. dr. E.C.M. van Gorp Prof. dr. A.J. Duits
Overige leden:
Prof. dr. P.M. van Hagen Prof. dr. P. Patka Prof. dr. A. Verbon Prof. dr. A.B.J. Groeneveld Prof. dr. D.P.M. Brandjes Prof. dr. T. van der Poll
Co-promotor: Dr. M.D. de Kruif
Contents Part I: Introduction 1. General outline of the thesis
9
2. Introduction and epidemiology of fever
13
3. Th e diagnostic role of procalcitonin and other biomarkers in discriminating infectious from non-infectious fever
23
Part II: Biomarkers 4. Th e acute phase response is not predictive for the development of arthritis in seropositive arthralgia – a prospective cohort study
41
5. A dditional value of procalcitonin for diagnosis of infection in patients with fever at the emergency department
51
6. P rocalcitonin as a potent marker of bacterial infection in febrile Afro-Caribbean patients at the emergency department
69
7. Procalcitonin in children with suspected novel influenza A (H1N1) infection
81
8. PTX3 predicts severe disease in febrile patients at the emergency department
91
Part III: Summary and appendices 9. Summary and discussion
107
10. List of publications
117
11. Nederlandse samenvatting en discussie
121
12. Dankwoord
131
13. Curriculum Vitae
135
Part I: Introduction
7
Chapter 1
General outline of the thesis
9
Chapter 1
T
his thesis aims to study biomarkers in inflammation and infection, with a special focus on the distinction between infectious and non-infectious fever. The thesis consists of three parts, part I being this introduction, in which the concept of fever in infectious and non-infectious disease is discussed. Furthermore, in this part we provide an overview of the epidemiology of febrile disease, as studied both in a general hospital in the Netherlands and in a general hospital in Curaçao. Also, a review of current literature on biological markers in non-infectious fever is given. Part II describes our clinical studies with focus on biomarkers in different cohorts of patients with infectious and non-infectious fever. In part III, we summarize the findings of this thesis and discuss future research.
Part I: Introduction In this part, a general introduction to the concept and pathophysiology of fever is given. To be able to discuss the diagnostic properties of biomarkers in inflammation and infection, it is of great importance to have good insight into the prevalence of febrile disease and diagnosis; the incidence and prevalence of a certain disease influence the diagnostic test properties, such as positive and negative predictive value. In this section, we describe the epidemiology and etiology of febrile disease in the emergency department (ED) both in the Netherlands and in Curaçao. As many cases of fever do not have an infectious etiology, we review current literature on biological markers as diagnostics in patients with non-infectious fever, with special attention to the biological marker procalcitonin (PCT).
Part II: Biomarkers This section focuses on biomarkers, suitable for discrimination between – bacterial - infection and non-infectious inflammation. As a model of non-infectious inflammation, we studied the prognostic value of biomarkers during the acute phase response in rheumatoid arthritis. We present two emergency department studies – one in Amsterdam and one in Curaçao – investigating the diagnostic properties of currently used and new biomarkers, such as PCT and the long pentraxin 3 (PTX3). In addition, we studied the diagnostic behavior of PCT in children with suspected H1N1-influenza during the 2009 pandemic.
Part III: Summary and appendices In this part, we summarize the most important findings of our studies and we discuss future research. Data of a prospective pilot study on the ED, using PCT as a guide for antibiotic therapy, are presented.
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Chapter 2
Introduction and epidemiology of fever Authors: M. Limper, A.J. Duits and E.C.M. van Gorp Partly published in Neth J Med. 2011 Mar; 69(3): 124-8
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Chapter 2
F
ever as a symptom of disease is an ancient concept, with the oldest known reference dating back to the sixth century BC, when a pictogram of a flaming brazier, symbolizing fever and the local warmth of inflammation was used in Sumerian cuneiform inscriptions1. Several centuries later, Hippocratic physicians proposed that body temperature and homeostasis in general was controlled by a delicate balance between four corporal humors - blood, phlegm, black bile and yellow bile -; fever was thought to be the result of an excess of yellow bile2. During the Middle Ages, demonic possession was believed to be causing fever. By the 18th century, after the development of improved microscopes by van Leeuwenhoek and the subsequent birth of microbiology, it was hypothesized that body heat was a result of fermentation and putrefaction in the blood3. In the late 19th century, metabolic bodily processes were recognized as the source of fever, as well as the fact that tight control of temperature is essential for general well-being4. Further research in the 20th century has shown that different endogenously produced molecules of leukocytic origin, such as interleukin 1 (IL-1), -2, 6, tumor necrosis factor (TNF) and interferons (IFN), act on the thermoregulatory center of the hypothalamus, thus eliciting fever5. Different definitions of fever for different purposes can be found. In clinical practice, a temperature greater than 38.0 ºC is considered fever, and fever is typically defined as a pyrogen-mediated rise in body temperature above this temperature. Physiologically, it has been defined as “a state of elevated core temperature, which is often, but not necessarily, part of the defensive responses of multicellular organisms (host) to the invasion of live (micro-organisms) or inanimate matter recognized as pathogenic or alien by the host”6. The elevated body temperature during fever should be distinguished from that occurring in hyperthermia. In fever, the rise in temperature is a result of well-controlled hypothalamic thermoregulation, whereas in hyperthermia the rise in body temperature is unregulated and pyrogenic cytokines are not directly involved, representing a failure of homeostasis7. The function of fever is still under debate, with different studies showing both potentiating and inhibitory effects of the response to infection. Phylogenetic studies have shown that fever is widespread within the animal kingdom; as the rise in temperature is metabolically expensive but still is well-preserved in evolution, it has been argued that fever has to be an adaptive and beneficial response8. Furthermore, animal studies have demonstrated enhanced resistance to infection during experimentally increased temperature7. In human in vivo studies associations between higher temperatures and better disease outcome have been observed9-11. On the contrary, it has been suggested that pyrogenic cytokines such as IL-1, IL-2 and TNF are involved in at least part of the local and systemic response to infection, with higher levels of circulating cytokines correlating with less favorable outcome. In experimental studies, the adverse effects of gram-negative sepsis or lipopolysaccharide injections were attenuated by pre-treating animals with IL-1 antagonists and anti-TNF antibodies12, 13.
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Introduction and epidemiology of fever
Body temperature is controlled deep in the hypothalamus, where thermosensitive receptors are affected by blood temperature, as well as via direct neural connections that measure warmth and cold in skin and muscle. In addition to the peripheral temperature input, the hypothalamus possesses an independent circadian temperature rhythm oscillating around a steady setpoint, unaffected by ambient temperature. The hypothalamus interprets peripheral temperature information and compares this with the independent circadian rhythm, resulting in peripheral heat conservation or loss of heat production. During fever, the hypothalamic setpoint is reset to a higher level while the thermoregulatory mechanisms are still maintained5. Substances causing fever, so-called pyrogens, can be divided into exogenous and endogenous pyrogens. Most exogenous pyrogens are microbes, toxins or other microbial products, either working directly on the hypothalamus or inducing the release of endogenous pyrogens, derived form the host’s cells. Some endogenously produced molecules are also capable of inducing endogenous pyrogens, such as antigen-antibody complexes and complement factors. Endogenous pyrogens, most importantly IL-1, IL-6, TNF-α and IFN-γ, interact with brain microglia and brain endothelial receptors, thus activating the arachidonic acid pathway. This in turn results in the production of cyclo-oxygenase derived prostaglandins, prostacyclins and thromboxane. Prostaglandin E2, most notably, increases the hypotha-
Figure 1: Simplified pathogenesis of fever
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Chapter 2
lamic thermostatic setpoint. As a result, efferent nerves are activated through sympathetic stimuli and vasoconstriction and heat conservation occurs5, 14, 15, 16. Febrile illness is worldwide a very frequent cause of attendance at emergency departments (EDs). Fever was the third reported complaint among most frequently reported specific principal reasons for visiting an ED in the United States in 2005, accounting for 4.4 - 7.5 % of all ED consultations and for up to 30 % in non-surgical patients17, 18. Although fever is often caused by bacterial, viral or parasitic infections, an elevated body temperature can be observed in many non-infectious diseases, such as auto-immune diseases, malignancies and thrombo-embolic processes. Distinction between causes of fever is clinically important, because infectious fever will be treated with antibiotics or antiviral medication, whereas non-infectious fever might be treated with immunosuppressive drugs. Although major breakthroughs have been observed in the treatment and prevention of infections over the past decades, tools for discriminating between infectious and noninfectious causes of fever – either physical examination or supplemental diagnostics - are still very inaccurate and non-specific19-21. In order to gain better insight in the epidemiology of fever at the first aid department, we investigated two patient cohorts with non-surgical fever, one at a general hospital in Amsterdam, the Netherlands, and one in a general hospital in Curaçao, a subtropical SouthAmerican island close to the coast of Venezuela. In both cohorts, as expected, most patients were diagnosed with a bacterial infection and were treated with antibiotics. Infection could be confirmed in approximately 40% of the patients; approximately 80% of these patients were diagnosed with a confirmed bacterial infection. Leading causes of bacterial infection were pneumonia and urinary tract infections. Approximately 5% of patients with confirmed infection had a viral disease; parasitic or fungal disease was diagnosed in 1.4% and 10.0%, respectively. Non-infectious fever was diagnosed in 4.2% en 5.2% of patients, respectively. Table 1: patient characteristics Slotervaart Hospital
n = 213 n (%) / median (IQR) Sex, female
111 (52.1)
Age, yrs.
66 (46 – 79)
Hospitalization
187 (87.8 %)
Admission to Intensive Care Unit
18 (8.5 %)
Mortality
9 (4.2 %)
IQR = interquartile range
Mortality in both cohorts was substantial, 4.2% and 7.9%, respectively. Patient characteristics are given in table 1 (Slotervaart Hospital, Amsterdam, the Netherlands) and table 2 (St. Elisabeth Hospital, Curaçao). Most reported diagnoses are given in figures 1 and 2.
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Introduction and epidemiology of fever Table 2: patient characteristics St. Elisabeth Hospital
n = 403 n (%) / median (IQR) Sex, female
196 (49)
Age, yrs.
52 (32 – 71)
Hospitalization
223 (55.6 %)
Admission to Intensive Care Unit
18 (4.5 %)
Mortality
32 (7.9 %)
IQR = interquartile range
Figure 2: Most reported final diagnosis of febrile patients at the Emergency Department of the Slotervaart Hospital, Amsterdam, the Netherlands, during the year 2008 (URTI: upper respiratory tract infection)
Figure 3: Most reported final diagnosis of febrile patients at the Emergency Department of the St. Elisabeth Hospital, Curaçao, during the year 2008 (UTI: urinary tract infection; NOS: not-otherwise specified)
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Chapter 2
These two epidemiological studies show that fever in patients at the ED is mainly caused by bacterial infection, but that a substantial part of patients suffer from a non-infectious febrile disease. Moreover, in these patient populations, high mortality numbers are observed. No data on incidence of non-infectious fever at the ED have been published before. The incidence of infectious fever we show, however, is comparable to that found in other studies17, 18; we assume that our findings can be extrapolated to other hospitals worldwide, particularly because, despite substantial differences in health care setting, population and climate, results are quite equal between the two investigated hospitals. The search for new and better biomarkers that predict the presence of bacterial infection in febrile patients has become increasingly relevant, particularly in view of rising antibiotic resistance and medical costs worldwide. Currently used biomarkers, such as C-reactive protein (CRP) have repeatedly been shown to be not sensitive or specific enough to discern between different causes of fever20. Recently, use of several new biomarkers for this purpose has been studied. Among many potential markers, procalcitonin (PCT) - a prohormone of calcitonin, under physiological conditions produced by the thyroid gland - appears to be a promising and specific marker of bacterial infection in different patient groups and conditions, varying from neonatal sepsis to outpatients with respiratory complaints. As has been repeatedly shown, procalcitonin has a high specificity (ranging from 90-98%) for predicting bacterial infections and would therefore, theoretically, be suitable for the discrimination between patients with bacterial fever and patients with underlying non-infectious febrile disease22-25.
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Introduction and epidemiology of fever
References 1. Majno, G. The Healing Hand: Man and Wound in the Ancient World. Cambridge, Mass: Harvard University Press; 1975:57 2. The Hippocratic Treatises, Diseases IV, VII 568-572; transl. IM Lonie. Berlin, Germany: De Gruyter; 1981:28 3. Atkins E. Fever: its history, cause and function. Yale J Biol Med. 1982;55:283-287 4. Bernard C. Leçons sur les Phénonèmes de la Vie communs aux Animaux et aux Végétaux. Paris, France: Baillière et fils; 1879 5. CA Dinarello, JG Cannon and SM Wolff. New concepts on the pathogenesis of fever. Rev Inf Dis 1988;1: 168-188 6. IUPS Thermal Commission. Glossary of terms for thermal physiology: second edition. Pflugers Arch. 1987; 410: 567-587 7. P. Mackowiak. Concepts of Fever. Arch Int Med 1998;158: 1870-1881 8. Kluger MJ, Ringler DH, Anver MR. Fever and survival. Science. 1975;188:166-168 9. Bryant RE, Hood AF, Hood CE, Koenig MG. Factors affecting mortality of gram-negative rod bacteremia. Arch Int Med. 1971; 127: 120-128 10. Weinstein MR, Iannini PB, Staton CW, Eichoff TC. Spontaneous bacterial peritonitis: a review of 28 cases with emphasis on improved survival and factors influencing prognosis 11. Dorn TF, DeAngelis c, Baumgardner RA, et al. Acetaminophen: more harm than good for chickenpox? J Pediatr. 1989; 114:1045-1048) 12. Ohlsson K, Bjork P, Bergenfeldt M, Hageman R, Thompson RC. Interleukin-1 receptor antagonist reduces mortality from endotoxin shock. Nature. 1990;348:550-552 13. Overbeek BP, Veringa EM. Role of antibodies and antibiotics in aerobic gram-negative septicemia: possible synergism between antimicrobial treatment and immunotherapy. Rev Infect Dus. 1991; 13:751-760 14. Dinarello CA. Infection, fever, and exogenous and endogenous pyrogens: some concepts have changed. J Endotoxin Res. 2004;10(4):201 15. Mackowiak PA, Wasserman SS, Levine MM. A critical appraisal of 98.6 degrees F, the upper limit of the normal body temperature, and other legacies of Carl Reinhold August Wunderlich. JAMA. 1992;268(12):1578 16. Atkins E. Pathogenesis of fever. Physiol Rev. 1960;40:580; Coceani F, Bishai I, Lees J, Sirko S. Prostaglandin E2 and fever: a continuing debate. Yale J Biol Med. 1986;59(2):169) 17. Nawar EW, Niska RW, Xu J. National Hospital Ambulatory Medical Care Survey: 2005 emergency department summary. Adv Data. 2007;1-32. 18. Shimoni Z, Niven M, Kama N, Dusseldorp N, Froom P. Increased complaints of fever in the emergency room can identify influenza epidemics. Eur J Intern Med. 2008;19:494-498 19. Marnell L, Mold C, Du Clos T W. C-reactive protein: ligands, receptors and role in inflammation. Clin Immunol 2005; 117: 104-111 20. Meisner M. Biomarkers of sepsis: clinically useful? Curr Opin Crit Care 2005; 11: 473-480 21. Pepys M B, Hirschfield G M. C-reactive protein and its role in the pathogenesis of myocardial infarction. Ital Heart J 2001; 2: 804-806 22. Chirouze C, Schuhmacher H, Rabaud C, Gil H, Khayat N, Estavoyer J M et al. Low serum procalcitonin level accurately predicts the absence of bacteremia in adult patients with acute fever. Clin Infect Dis 2002; 35: 156-161
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Chapter 2 23. Briel M, Schuetz P, Mueller B, Young J, Schild U, Nusbaumer C et al. Procalcitonin-guided antibiotic use vs a standard approach for acute respiratory tract infections in primary care. Arch Intern Med 2008; 168: 2000-2007 24. Uzzan B, Cohen R, Nicolas P, Cucherat M, Perretn G Y. Procalcitonin as a diagnostic test for sepsis in critically ill adults and after surgery or trauma: a systematic review and meta-analysis. Crit Care Med 2006; 34: 1996-2007 25. Assicot M, Gendrel D, Carsin H, Raymond J, Guilbaud J, Bohuon C et al. High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 1993; 341: 515-518
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Chapter 3
The diagnostic role of procalcitonin and other biomarkers in discriminating infectious from non-infectious fever Authors: M. Limper, MD; M.D. de Kruif, MD, PhD; A.J. Duits, PhD; D.P.M. Brandjes, MD, PhD; E.C.M. van Gorp, MD, PhD Published in: J Infect. 2010 Jun; 60(6): 409-16
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Procalcitonin in discriminating infectious from non-infectious fever
Abstract Fever is not only observed in the course of a bacterial or viral infection, but can be a symptom of, for instance, auto-immune, malignant or thromboembolic disease. Determining the etiology of fever in a fast and reliable way is of pivotal importance, as different causes of fever may ask for different therapies. Neither clinical signs and symptoms, nor traditional biomarkers, such as CRP, leukocytes and ESR have sufficient sensitivity and specificity to guide treatment decisions. In this review we focus on the value of traditional and newer biomarkers in non-infectious febrile diseases. Procalcitonin (PCT) seems to be the most helpful laboratory marker for the differentiation of causes of fever, particularly in autoimmune, autoinflammatory and malignant diseases. Keywords: fever, biological markers, autoimmune diseases, C-reactive protein, procalcitonin.
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Chapter 3
Introduction Fever is one of the most frequent symptoms seen in patients by both family doctors and in the emergency departments of hospitals. Fever is the third leading cause of visits to an emergency department in the United States; approximately 10% of emergency patients are prescribed antibiotics1. Besides infections - bacterial, viral or parasitic – there are several non-infectious medical conditions that can cause an elevated body temperature. Fever is observed in systemic diseases such as systemic lupus erythematosus and rheumatoid arthritis, in inflammatory bowel diseases, in auto-inflammatory syndromes, as a paraneoplastic phenomenon in malignancy or during neutropenia after chemotherapy, as a result of tissue loss in ischemic or thromboembolic processes, in endocrine disorders and as a result of medication. During the past 10 years, the interest in biomarkers that discriminate between infectious and non-infectious causes of fever has grown. Moreover, the need for new and better biomarkers that predict the presence of bacterial infection in febrile patients has become increasingly relevant. ESR has now been used clinically for almost 90 years, whereas C-reactive protein (CRP) has been used routinely for the past 30. It has repeatedly been shown that the clinically often-used CRP is part of an intrinsically, non-specific acute phase reaction and is therefore not sensitive or specific enough to discern between different causes of fever. The same holds true for other traditional biomarkers. Although major breakthroughs have been observed in the treatment (antibiotics) and prevention (vaccines) of infections during the past decades, tools for discriminating between infectious and non-infectious causes of fever are still very inaccurate and non-specific2-4. When treatment is started, currently used laboratory markers are of limited value regarding the guidance of treatment. Finally, a new generation of biomarkers is on the verge of clinical introduction.
Figure 1: Time course of induction of various parameters of the systemic inflammatory system after stimulus (thoracic surgery). Concentrations are relative and adapted to the Y-axis. Figure courtesy of M. Meisner, 1999, adjusted with permission.
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Procalcitonin in discriminating infectious from non-infectious fever
Discriminating between an infectious or non-infectious origin of a fever is of major importance, because the former can be treated with antibiotics, whereas the latter might call for strong, immunosuppressive drugs. An ideal biomarker would have a sensitivity and specificity of 100%, providing the treating physician with certain knowledge about the cause of the fever. Recently, several studies focusing on new markers, have been undertaken. Procalcitonin (PCT) is such a new marker, about which a number of clinical studies have been published. PCT is a specific marker of bacterial infection in different patient groups and conditions, varying from neonatal sepsis to outpatients with respiratory complaints5-12. After a bacterial stimulus in healthy volunteers, PCT levels rise within 4 hours, reaching peak levels after 6 hours and maintaining a plateau through 8 and 24 hours13 (figure 1). PCT has a half-life time of approximately 24 hours, independent of renal function14. As has been repeatedly shown, procalcitonin in general has a high specificity (ranging from 90-98%) for bacterial infections 7, 8, 11, 15 and would therefore, theoretically, be suitable for the discrimination of bacterial fever in patients with underlying non-infectious febrile disease. However, slightly elevated PCT levels can be observed 3-24 hours after aerobic exercise16 and not all bacterial infections give rise to elevated PCT levels5. Moreover, although the negative cut-off value for PCT is generally believed to be < 0.5 ng/mL, optimal cut-off points vary between different studies and patient populations. A mild increase of PCT values could be observed in some patients with an inflammatory, non-infectious condition17. The general departure point in the majority of earlier studies, that focus on circulating levels of biomarkers in febrile patients, is infection. In this review we describe, for the first time, the diagnostic applicability of traditional laboratory markers, procalcitonin and other promising biomarkers, taking non-infectious febrile diseases as a departure point. It is particularly within this interesting subgroup of fever patients, who, as a result of the lack of objective parameters to guide the prescribed treatment, are likely to receive antibiotics if the treating physician has reason to doubt the etiology of the fever. Biomarkers with a higher test sensitivity and specificity, regarding the discrimination between fevers with infectious and non-infectious causes, will reduce the amount of antibiotic prescriptions in this patient group and thus lead to a reduction in costs and the development of resistant bacterial strains. The distinction between viral and bacterial infection, as well as fevers caused by the use of medication, are beyond the scope of this article. Search terms were “Biological markers [MeSH]”, “Fever [MeSH]”, “C-reactive protein [MeSH]”, “Blood sedimentation [MeSH]”, “Procalcitonin [MeSH]”, “Serum Amyloid Protein A [MeSH]”, “PTX3 Protein [MeSH]”, “Autoimmune Diseases [MeSH]”, “Inflammatory Bowel Diseases [MeSH]”, “Periodic fever syndromes”, “Neoplasms [MeSH]”, “Neutropenia [MeSH]”, “Ischemia”, “Thrombo-embolism”, “Endocrine System Diseases [MeSH]”, “Diagnosis” and “NOT infection”. Non-English articles and non-human studies were excluded from this review.
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Chapter 3
Autoimmune and non-infectious inflammatory diseases Autoimmune disease is defined as a disease with proof of direct transmissibility of the characteristic lesions of the disease from human to human or from human to animal – i.e., demonstrating the direct pathological effect of autoantibodies -; by indirect re-creation of human disease in an animal model; or by circumstantial evidence such as a positive family history, presence of other autoimmune phenomena in the same patient, infiltrating mononuclear cells in affected tissue, preferential use of certain MHC-II alleles, high serum levels of IgG autoantibodies, deposition of antigen-antibody complexes in affected tissue and/or improvement of symptoms with immunosuppressive therapy18. However, in many diseases it remains difficult to prove autoimmunity. Ongoing research has led to the insight that in some of the diseases that were originally categorized as autoimmune diseases – for instance, sarcoidosis, Behcet’s disease and temporal arteritis -, the role of auto-antibodies in the pathophysiology was overestimated. These disorders are nowadays coined as non-infectious inflammatory syndromes, with the autoinflammatory disorders as a well-circumscribed subgroup. Patients suffering from systemic diseases such as rheumatoid arthritis, systemic lupus erythematosus or vasculitides are prone to infection, particularly during immunosuppressive therapy19. As most auto-immune and non-infectious inflammatory diseases are characterized by fever, the differentiation of infection from active systemic disease is often difficult, but it has important clinical consequences. For decades, rheumatologists have been using the erythrocyte sedimentation rate (ESR) as an indicator of activity of autoimmune disease and infection. However, during the past 20 or so years, the value of using ESR has been questioned, as sensitivity and specificity numbers vary strongly between different studies and diseases. ESR is not helpful in discriminating between active autoimmune disease and infection20. The only reliable use of ESR seems to be in the diagnostic process of temporal arteritis, where a low ESR gives a high negative predicting value21. CRP, although widely used as an early and sensitive marker of infection and inflammation, is not sensitive enough to discriminate between infectious episodes or the exacerbation of an underlying autoimmune disease. Although values of CRP are generally intermediate to low in patients with active SLE, it is not possible to differentiate between SLE activity and infection as a cause of fever, based on CRP alone22-24. Patients with SLE and serositis show elevated CRP levels. However, elevated CRP levels in SLE patients without clinical signs of serositis are generally assumed to be indicative of bacterial infection25. Persistent elevations of circulating IL-6 levels have been shown to be associated with outcome in different patient groups26. However, despite fast and relatively high peak values during acute phase response, interleukin-6 (IL-6) has no additional value over CRP in discriminating systemic infection from absence of infection in patients with SLE, as plasma IL-6 concentrations are elevated in a significant amount of patients with autoimmune diseases in the absence of infection24.
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Procalcitonin in discriminating infectious from non-infectious fever
Serum amyloid A (SAA) has the same initial kinetics as CRP in the acute phase response, with a less rapid decrease after initial elevation. Elevated serum levels of SAA have been described in a wide variety of diseases, including rheumatoid arthritis, ankylosing spondylitis, Behcet’s syndrome and Crohn’s disease27. No studies using SAA as a marker for distinction between infectious and non-infectious fever have been undertaken. Due to the shared acute phase pattern with CRP and its longer half life, it is not to be expected that SAA is a helpful marker for this purpose. SAA may be useful in the evaluation of therapy response and the determination of progression to amyloidosis in these patient groups28-30. PCT seems to be a promising marker in differentiating between autoimmune-induced fever and infectious-bacterial fever. Strongly elevated PCT levels have been observed in several systemic and localized bacterial infections5, 6, 15, 24. PCT appears to have a higher specificity for bacterial infection than other markers, although it is apparent that not all patients with bacterial infection have elevated PCT levels5. In a cohort of patients with Wegener’s granulomatosis, elevated PCT levels were described in those suffering from bacterial infection, whereas PCT levels were normal in patients with active autoimmune disease. In the same cohort, CRP could not be used for differentiation purposes23. In a study in patients with active Behcet’s disease, PCT values were not elevated, whereas CRP and IL-6 levels were higher, when compared to healthy controls31. PCT values in patients with diverse systemic autoimmune diseases accompanied by infectious fever were elevated, when compared to values in patients with systemic autoimmune diseases and noninfectious fever32. Circulating PCT levels in patients with early RA and reactive arthritis were shown to be normal, whereas PCT levels in patients with bacterial sepsis were elevated 33. Recent preclinical in vitro studies focus on glyco-biomarkers and vascular endothelian growth factor (VEGF). In rheumatoid arthritis (RA), glyco-biomarkers have been proposed as future markers for diagnosis and prognosis. Glyco-biomarkers are sugar molecules that are an integral feature of nearly all biomolecules; they may represent a way in which immunotolerance can be bypassed. IgG-G0 (IgG-agalactosyl) may be a diagnostic and prognostic marker in RA, with levels of IgG-G0 exhibiting a correlation with the severity and duration of the disease. The relationship between glycosylation and infection still has to be elucidated34. Serum levels of VEGF correlate well with RA disease severity and swollen joint counts. VEGF might play an important part in the establishment and promotion of RA and might serve as a marker for disease activity, with a more active disease being associated with higher serum levels35.
Autoinflammatory diseases The autoinflammatory diseases are a heterogeneous group of hereditary syndromes characterized by seemingly unprovoked periodic episodes of inflammation without high titers of autoantibodies or antigenspecific T lymphocytes and in the absence of infection36. A central role for proteins with a PYRIN domain has been suggested. In normal states, pyrin
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Chapter 3
downregulates activity of a caspase recruitment domain which promotes elevated levels of IL-Iβ and NF-κB. In autoinflammatory disease, this downregulation is affected, resulting in the production of IL-Iβ and eventual inflammation36. Major autoinflammatory syndromes are familial Mediterranean fever (FMF), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) and tumor necrosis factor receptor superfamily 1A-associated periodic syndrome (TRAPS). Adult onset Still’s disease, although in contrast with other autoinflammatory syndromes non-hereditary, associated with longer exacerbations and with possible bone and cartilage destruction, may be classified as an autoinflammatory syndrome as it leads to comparable clinical features, in the absence of evidence for a link to microorganisms or circulating auto-antibodies37. Similar to other chronic inflammatory diseases, yet significantly more prevalent, the most severe complication of autoinflammatory syndromes is amyloidosis, leading to end-stage renal failure38. Susceptibility genes in most of the autoinflammatory diseases have been identified and insight in molecular pathogenesis has improved. This has led to better diagnosis and more rational therapy, with a growing interest in biological therapy with anti-TNF and anti-IL-I agents39. However, only a few studies have focused on the acute phase response during exacerbations of autoinflammatory diseases, with evidence for marked elevations of CRP, the long pentraxin 3 (PTX3), serum amyloid A protein and IgD levels40-42. Attacks of FMF can be severe and may mimic acute abdominal syndromes such as appendicitis or pelvic inflammatory disease so closely, that abdominal surgery can be mistakenly performed. A major challenge in the treatment of FMF patients, in particular, is to distinguish FMF attacks from infectious disease. One study showed that levels of CRP and PTX3 were increased during an FMF attack, whereas PCT was elevated above normal cutoff values in only 2 out of 22 patients43. Thus, PCT may be a promising marker to distinguish infectious disease from sterile FMF attacks. In a study comparing PCT values in patients with adult onset Still’s disease with and without infectious fever, high levels of PCT were also observed in patients without bacterial infection, indicating that PCT is not useful for the differentiation between exacerbations of Still’s disease and infection32. High levels of serum ferritin are a common finding in Still’s disease. Glycosylated ferritin, in non-pathological conditions representing more than half of the total serum ferritin, is often very low (< 20%) in adult onset Still’s disease, while the level is higher (20-50%) in other inflammatory conditions. However, also during severe systemic infections, glycosylated ferritin levels < 20% can be found44.
Inflammatory bowel diseases The two major inflammatory bowel diseases (IBD) are ulcerative colitis and Crohn’s disease. Both diseases are treated with corticosteroids and immune modifying agents, resulting in a relatively immune compromised state. Patients with IBD are particularly susceptible to Clostridium difficile infection and have a four times higher chance of dying as a result of infection than patients without IBD45. The pathogenesis of IBD has still to be elucidated. A cascade of immunologic events occurs, resulting in increased local release of pro-inflammatory cytokines in the gut. Il-
29
Procalcitonin in discriminating infectious from non-infectious fever
6, IL-10 and TNF-α, in particular, have been shown to play a pivotal role in the chronic inflammatory process46, 47. As ulcerative colitis and Crohn’s disease are different disease entities both clinically and pathophysiologically, different acute phase responses during exacerbations can be expected. However, the scarce research addressing the differentiation between exacerbations of IBD and infectious colitis does not focus on ulcerative colitis and Crohn’s disease separately. Hence, in this review we are forced to consider the two diseases as one group. As diverse cytokines are produced in large amounts during exacerbations of IBD, levels of regularly used laboratory markers such as CRP or ESR will be elevated, and they therefore possess little discriminatory value48. However, an accumulation of evidence points towards an association between oxidative stress and exacerbations. Markers for lipid peroxidation and antioxidant status have been shown to be higher in patients with active IBD, indicating enhanced oxidative stress and decreased anti-oxidant status49. There is a proven correlation between oxidative stress and the severity of sepsis; in non-septic infections the levels of oxidative stress should be low50. Emergency patients with bacterial infection were shown to have normal markers for oxidative stress51. A combination of elevated acute phase proteins and normal markers for oxidative stress suggests the presence of infection in patients with IBD, whereas an exacerbation of IBD might be indicated by elevated acute phase proteins and markers for oxidative stress. As a complicating factor, patients suffering from Chronic Granulomatous Disease (CGD) - a rare disorder caused by defects in the phagocyte NADPH oxidase resulting in the inability to generate reactive oxygen intermediates - may present with a clinical picture mimicking Crohn’s disease52, which makes a pathogenic role for oxidative stress less likely. One study assessed the value of PCT in a cohort of 51 IBD patients and 25 patients with self-limiting colitis. All IBD patients exhibited low levels of PCT in serum, independent of disease activity, whereas patients with infectious colitis were shown to have elevated PCT values. In this study the positive, predictive value for diagnosing infectious colitis was 96%, with a negative predictive value of 93%53.
Malignant disorders Neoplasms contribute to between 7-20% of the cases of fever of unknown origin54, 55. Fever is particularly associated with non-Hodgkin lymphoma, leukemia, renal cell carcinoma and hepatocellular carcinoma. Although the pathophysiology of neoplastic fever is not well understood, it is suspected to be cytokine mediated, with emphasis on IL-1, IL-6, TNF-α and interferon. It has been suggested that tumor cells produce pyrogens; another proposed explanation for neoplastic fever is the release of TNF by necrotic bone marrow. In neutropenic patients receiving chemotherapy, the discrimination between neoplastic fever and infectious fever is essential55. The value of CRP in differentiating infectious from non-infectious fever in neutropenic patients has been studied extensively. It has repeatedly been shown that CRP is an unreliable marker of bacterial infection in this patient group56-58.
30
Chapter 3
A recent systematic review identifies PCT as a valuable tool in determining the etiology of fever in neutropenic patients, with elevated levels of circulating PCT in patients with bacterial fever10. One study comparing the value of PCT, neopterin, CRP, IL-6 and IL-8 as diagnostic markers for this purpose showed a high, negative predictive value of PCT for Gram-negative bacteremia and non-significant results for the other markers9. It has been argued that a risk-assessment model, including clinical parameters together with laboratory markers such as PCT or IL-8, should be constructed, thus enabling physicians to accurately define a low-risk group of febrile neutropenic patients59.
Ischemic diseases During the first days after myocardial fever, low grade fever is a common observation. A relationship between the extent of necrosis and the rise in temperature has been shown60, 61. Animal studies indicate that elevated corporal temperature might be harmful prior to and directly after an acute coronary syndrome or myocardial infarction62. Fever after myocardial tissue loss occurs due to ischemic cell death, resulting in the production of cytokines. TNF, a very potent inflammatory mediator, has been shown to be present to an excessive degree in plasma during this process63. In patients who have suffered an acute stroke, fever is also a common observation. Although approximately 25% of patients who have suffered an acute stroke and fever suffer from concomitant infection, about 15% of patients have a fever without a recorded infection. Fever in this patient group is associated with poor outcome and severe cerebrovascular damage. A correlation between cerebral tissue loss and fever has been noted. The only discriminating characteristic between the infectious and non-infectious causes of fever is the early onset of fever in non-infectious patients64, 65. No clinical studies, investigating the role of biomarkers in differentiating between infectious and non-infectious etiology of fever in patients suffering from myocardial infarction or acute stroke, have been undertaken. Fever is observed in a substantial number of patients with pulmonary embolism (PE). The exact etiology of this fever is unclear. However, similar cytokine cascades as in true ischemic conditions may be expected66. Based on ESR and leukocyte counts, no differentiation between PE and community acquired pneumonia can be made67. One study assessed the value of PCT in patients with possible PE. Out of 40 patients with fever and clinical and radiological findings consistent with pleuritis or pneumonia, 10 patients were diagnosed with PE and 30 with pneumonia. But while high CRP levels were measured in these 10 patients, PCT levels were normal68. These findings need to be confirmed within the context of a larger trial.
Endocrine disorders Phaeochromocytoma is classically associated with spiking fever. In some phaeochromocytomas, a central role for IL-6 can be observed. Based on laboratory markers, phaeochromocytoma may be difficult to distinguish from an abscess, for instance, as the elevation of IL-6
31
Procalcitonin in discriminating infectious from non-infectious fever
is followed by a general inflammatory reaction. High levels of leukocytes, fibrinogen and CRP will be observed69. In acute adrenal insufficiency, fever is one of many aspecific findings. It is unclear whether this fever is indicative of general inflammation or whether it is due to the precipitating infection that is most often the direct cause of adrenal insufficiency70. As most cases occur after infection, and patients are likely to develop severe shock, initial empirical antibiotic treatment is warranted. No studies that try to discriminate between infectious and non-infectious causes of acute adrenal insufficiency have been undertaken. This is because treatment should not be delayed while diagnostic tests are performed. To our knowledge, procalcitonin has not been evaluated in endocrine disorders.
Conclusion In this review we have described the value of commonly used biomarkers, procalcitonin, and other experimental markers in patients with non-infectious fever. Fever is a very common symptom of many diseases, reflecting general inflammatory processes in the body that are not necessarily caused by infection. The proper treatment of febrile patients requires adequate and quick diagnoses. Laboratory markers with high sensitivity and specificity regarding the differentiation between infectious and non-infectious causes of fever may
Table 1: Differential diagnosis of non-infectious febrile diseases and relative values of CRP and PCT during steady state, exacerbation of underlying disease and bacterial infection (no change from baseline indicated by “=”, relative elevation from baseline indicated by “↑” or “↑↑”, insufficient data indicated by “??”)
Steady state Auto-immune/systemic · RA · SLE · Arteriitis temporalis
Exacerbation
Bact. infection
CRP
PCT
CRP
PCT
CRP
PCT
=
=
↑
=
↑↑
↑↑
=
=
=/↑
=
↑↑
↑↑
↑/↑↑
=
n/a
n/a
n/a
n/a ↑/↑↑
· Vasculitis other
=/↑
=
=/↑↑
=
↑/↑↑
· Sarcoidosis
=/↑
??
=/↑
??
↑/↑↑
??
=
=
↑
=
↑/↑↑
↑/↑↑
· Behcet’s Auto-inflammatory · FMF
=
=
↑↑
=/↑
↑/↑↑
↑/↑↑
· TRAPS/HIDS
=
??
↑↑
??
??
??
· Still’s disease
=
=
↑↑
↑↑
↑/↑↑
↑/↑↑
=
=
↑/↑↑
=
↑/↑↑
↑/↑↑
=
=
↑/↑↑
=
↑/↑↑
↑/↑↑
=/↑↑
=
n/a
n/a
=/↑↑
↑/↑↑
IBD · Crohn’s disease · Colitis ulcerosa Malignancy Tissue loss/ ischemia Endocrine
32
??
??
??
??
??
??
??
??
??
??
??
??
Chapter 3
support the treating physician in deciding to withhold the prescription of antibiotics, thus leading to cost reductions and the decrease of bacterial resistance. Traditional biomarkers, such as CRP, leukocytes and ESR do not have sufficient sensitivity and specificity to guide treatment decisions. To date, PCT seems to be the most helpful laboratory marker for this purpose, particularly in autoimmune, autoinflammatory and malignant diseases. PCT is not useful for the differentiation between exacerbations of Still’s disease and infection (Table 1). PCT has recently been introduced as a clinical marker in various hospitals all over the world, and is more and more accepted as a useful marker in the diagnostic process in febrile patients. However, the optimal cut-off values for PCT in different patient groups with different pathology still have to be constructed. At this moment, the PCT assay is still costly with a range of $ 20,- to $ 40,- per measurement, which makes it less suitable for resource-poor countries. Other novel biomarkers, such as SAA and PTX3, are still experimental; further research on these markers is required to determine their clinical value. It should be clear that no single biomarker is sensitive or specific enough to be relied on completely. Therapy decisions should still be based on a combination of medical history, physical examination and adjunctive tests. In specific cases, specific biomarkers can be used to discriminate infectious from non-infectious fever. Further research in specific patient groups should focus on integrating biological markers and clinical parameters into decision rules. As the usefulness of laboratory markers varies within different diseases, decision rules applicable to specific settings and diseases have to be developed. Despite the added value of procalcitonin and other “new kids on the block”, medicine still remains the science of uncertainty and the art of probability.
33
Procalcitonin in discriminating infectious from non-infectious fever
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37
Part II: Biomarkers
39
Chapter 4
The acute phase response is not predictive for the development of arthritis in seropositive arthralgia – a prospective cohort study Authors: M. Limper, MD*, L.A. van de Stadt, MD*, W.H. Bos, MD, PhD, M.D. de Kruif, MD, PhD, C.A. Spek, PhD, G. Wolbink, MD, PhD, D. van Schaardenburg, MD, PhD, E.C.M. van Gorp, MD, PhD * The first two authors contributed equally to this work Published in J Rheumatol. 2012 Oct; 39(10): 1914-7
41
The acute phase response is not predictive for rheumatoid arthritis
Abstract
Introduction In clinically active rheumatoid arthritis (RA) acute phase reactants such as C-reactive protein (CRP) can be elevated. Patients presenting with arthralgia and a positive test for anti-cyclic citrullinated peptide antibodies (aCCP) and/or IgM rheumatoid factor (IgM-RF) are at risk for developing RA. Elevated acute phase reactants in this phase could be predictive for the development of arthritis.
Methods 137 aCCP and/or IgM-RF positive patients were included. Patients were followed annually for the development of arthritis, which was defined as presence of one or more swollen joints at clinical examination. High sensitive CRP (hsCRP), Procalcitonin (PCT), SPLA2, Tumor necrosis factor (TNF)-α, IL-6, IL-12p70, IL-10 and interferon (IFN)-γ were measured in baseline serum samples. Gene expression focusing on a predefined panel of genes coding for inflammatory molecules was measured by means of multiplex ligation-dependent probe amplification (MLPA).
Results 35 patients (26 %) developed arthritis within a median time of 11 months (IQR 3.7 – 18 months). Circulating levels of cytokines, sPLA2, hsCRP and PCT were not different between patients with progression to clinical arthritis and patients without progression. However, a trend for IL12p70, TNF-α, IL-10, IL-6 and sPLA2 could be observed. No significant correlation between mRNA expression levels of inflammatory genes was found. Subgroup analysis of patients with early progression to arthritis showed significantly higher levels of mRNA expression of PARN and BMI1 as compared to patients without progression to arthritis.
Conclusions Although low grade inflammation is present before the onset of clinical arthritis in large cohorts and can be detected using consecutive measurements, a single measurement of acute phase reactants seems to be of limited value for prediction of development of arthritis in individual patients.
42
Chapter 4
Introduction Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease, of which the exact etiology remains to be elucidated. In the pathophysiology of RA, inflamed synovia play a central role, exhibiting varying changes with disease progression. In early, pre-clinical disease, hyperplasia and edema of synovial lining can already be observed. The synovial sublining is infiltrated by mononuclear cells, mainly macrophages and monocytes, T cells and B cells, producing cytokines and chemokines including IL-1, IL-6 and TNF-α. In clinically active RA, inflammation is represented in the blood by elevated levels of acute phase reactants such as high sensitivity C-reactive protein (hsCRP), which can thereby be used as a diagnostic marker for disease activity. Approximately half of patients with RA have specific serologic abnormalities several years before the onset of symptoms. The presence of an elevated serum level of IgMRheumatoid factor (IgM-RF) or anti citrullinated peptide antibodies (ACPA) in a healthy individual implies an increased risk for the development of RA1. Furthermore, a rise in CRP and secretory phospholipase A2 (SPLA2) levels has been associated with the development of RA2. However, it remains unclear whether these serological markers can be used to predict the development of arthritis prior to disease onset. This study was conducted to test the hypothesis that acute phase proteins can be used as markers to predict the development of RA in patients at increased risk.
Patients and methods
Study population The study was conducted at the Jan van Breemen Research Institute, Reade, Amsterdam. A detailed description of the study population can be found in previous publications3, 4. In short, patients with arthralgia and a positive anti-CCP2 and/or IgM-Rheumatoid Factor (IgM-RF) status were recruited. At the first visit, a trained investigator completed a questionnaire regarding the presenting symptoms. The absence of arthritis was confirmed by two independent investigators (WB or LAS and DS) of which one was a senior rheumatologist blinded for antibody status and medical history. Patients were excluded if one or both investigators observed any swollen joint and/or if chart review revealed past arthritis observed by a rheumatologist. Furthermore, patients previously treated with a disease modifying anti-rheumatic drug (DMARD) and patients with systemic lupus erythematosus or Sjögren’s syndrome were excluded due to the possibility of false-positive RF in these patients. During yearly follow-up visits, development of arthritis was independently confirmed by two investigators (WB or LAS and DS). Extra visits were planned if arthritis developed. Median follow-up was 21 months (range 6–48 months). Analysis for the current study started after inclusion of the first 137 consecutive patients from this cohort, recruited between September 2004 and November 2007.
43
The acute phase response is not predictive for rheumatoid arthritis
Furthermore, 20 patients with RA fulfilling the 1987 ACR criteria were selected as a positive control for hsCRP and SPLA2 measurements and 40 healthy blood bank donors were used as negative control.
Measurements Blood samples were obtained by venapuncture. RNA was isolated from PAXgenetm tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) containing RNA stabilizing buffer according to protocol and gene expression was measured by means of multiplex ligationdependent probe amplification (MLPA), focusing on a predefined panel of genes coding for inflammatory molecules, as described previously5. EDTA anticoagulated plasma was aliquoted and stored at -80°C. The blood was routinely screened for haematological and biochemical variables. hsCRP was measured using a highly sensitive latex-enhanced assay on a Hitachi 911 analyzer (Roche Diagnostics), according to the manufacturer’s instructions. Procalcitonin (PCT) levels were measured by a chemiluminescence sandwich immunoassay (BRAHMS AG, Hennigsdorf, Germany) as previously described6. SPLA2 levels were measured by ELISA as previously described7. Tumor necrosis factor (TNF)-α, IL-6, IL-12p70, IL-10 and interferon (IFN)-γ were measured by cytometric bead array (CBA) multiplex assay (BD Biosciences, San Jose, CA, USA). All samples were measured in a blinded fashion without knowledge of the clinical status of the patients.
Statistical Analysis Baseline demographic and clinical variables are tabulated and descriptive statistics are presented as numbers with percentages, medians with inter quartile ranges (IQR) or means with standard deviations when a normal distribution could be assumed. Differences in marker levels between groups were determined with an unpaired T-test. Correlations were determined by means of logistic regression analysis. CBA, MLPA and biomarker levels were transformed with their natural logarithm prior to statistical analysis to normalize the data. Subgroup analysis comparing early progressors, defined as patients with development of arthritis within the first IQR after inclusion in the study, with non-progressors was performed for all markers. A two-tailed p-value