Diagnostic and Prognostic Value of Hormokines as Biomarkers in Severe Infections

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as Biomarkers in Severe Infections

M. Christ-Crain and B. Müller

Introduction: What are ‘Hormokines’?

The term “hormokine” was proposed to encompass the cytokine-like behavior of hormones, during inflammation and infections [1]. The concept was based on the finding of a ubiquitous expression of calcitonin peptides (i.e., procalcitonin [PCT], calictonin gene related peptides (CGRPs), and adrenomedullin [ADM]) during sepsis. Calcitonin gene products are prototypes of hormokine mediators and can follow either a classical hormonal expression in neuro-endocrine cells or a cytokine-like ubiquitous expression pathway in various cell types [1]. The inflammatory release of hormokines can be induced either directly via microbial toxins (e.g., endotoxin) or indirectly via a humoral or cell-mediated host response (e.g., interleukin [IL]-1q , tumor necrosis factor [TNF]-[ , IL-6). Parenchymal cells (including liver, kidney, adi- pocytes, and muscle) provide the largest tissue mass and principal source of circulating hormokines in sepsis [2]. The greater mRNA induction and peptide release from parenchymal cells in comparison to circulating cells, appears to indicate a tissue-based, rather than a leukocyte-based mechanism of host defense, which is char- acteristic of hormokines. Lowering of circulating hormokines by specific antibodies improves survival in various animal models of mono- and polymicrobial sepsis.

This important finding suggests potential therapeutic use.

Calcitonin Peptides are Prototypes of Hormokines

PCT is a precursor peptide from the hormone calcitonin, and is also referred to as the prototype “hormokine” mediator. After translation from calcitonin-messenger RNA (mRNA), PCT is cleaved enzymatically into smaller peptides, finally to yield the thirty-two amino acid mature calcitonin [3]. In the traditional endocrine view, mature calcitonin is produced mostly in neuro-endocrine C-cells of the thyroid. In the absence of infection, the extra-thyroidal transcription of the calcitonin (CALC)-I gene is suppressed and is restricted to a selective expression in neuro-endocrine cells found mainly in the thyroid and lung. In these neuroendocrine cells, the mature hormone is processed and stored in secretory granules [2, 4].

Interestingly, a microbial infection induces a ubiquitous increase in CALC-I gene- expression and a constitutive release of PCT from all parenchymal tissues and differ- entiated cell types throughout the body [1]. The induction can be attenuated by cytokines also released during a viral infection (e.g., interferon [IFN]-* ). Thus, under septic circumstances, the entire body could be viewed as being an endocrine gland. Indeed, the transcriptional expression of calcitonin-mRNA is more uniformly

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upregulated in sepsis than are the mRNAs of the classical cytokines [5]. Accordingly, PCT levels increase several thousand-fold in severe infections, e.g., sepsis, with a superior diagnostic accuracy as compared to other parameters of microbial infections.

Calcitonin Peptides for the Improved Diagnostic Assessment of Severe Infections

The traditional clinical signs of infection and the routine laboratory tests in sepsis (e.g., C-reactive protein [CRP] or white blood cell [WBC] count) lack diagnostic accuracy and are sometimes misleading. In severe infection, most classical pro- inflammatory cytokines (e.g., TNF-[ , IL-1 q or IL-6) are increased only briefly or intermittently, if at all. Mortality in sepsis remains high, often due to delayed diagnosis and treatment. In view of this diagnostic and therapeutic dilemma, a more unequivocal test for the differential diagnosis of infection and sepsis is of para- mount importance. Recently, in an attempt to improve current definitions of the systemic inflammatory response syndrome (SIRS) and sepsis, it was suggested that PCT be included as an additional diagnostic tool to facilitate and expedite the difficult clinical diagnosis. This suggestion was based on evidence from the literature that, in sepsis, PCT levels increase several-fold until several thousand-fold and on admission this increase often correlates with the severity of the condition and with subsequent mortality [6]. A variety of studies and reviews have shown the superior diagnostic accuracy of PCT as compared to other parameters for the diagnosis of sepsis, inde- pendent of the origin of infection (references in [6]). Whereas the increase of other inflammatory markers, such as CRP, is attenuated by immunosuppressive medication (namely steroids), the diagnostic accuracy of PCT remains unaffected [7]. In addition, PCT seems to have a slight advantage over CRP because of its earlier increase upon infection and a better negative predictive value, as for example shown in children with fever of unknown origin [8].

The most frequent source of systemic infection is the lung with respiratory tract infections [6]. A common problem in clinical practice is that in respiratory tract infections, signs and symptoms of bacterial and viral infections overlap consider- ably. After obtaining the medical history, physical examination, laboratory results, and chest X-ray, the clinician is often left with considerable diagnostic uncertainty.

In view of this diagnostic and therapeutic dilemma, a more unequivocal test for the differential diagnosis of respiratory tract infections is clearly needed.

Calcitonin Peptides for Antibiotic Stewardship of Respiratory Tract Infections

The most frequent source of systemic infections is the lung [6]. Lower respiratory tract infections (LRTI), i.e., acute bronchitis, acute exacerbations of chronic obstruc- tive pulmonary disease (COPD) or asthma, and pneumonia, account for almost 10 % of the worldwide burden of morbidity and mortality. As many as 75 % of all antibiotic doses are prescribed for acute respiratory tract infections in spite of their pre- dominantly viral etiology. This excessive use of antibiotics is the main cause of the spread of antibiotic-resistant bacteria [9, 10]. Thus, decreasing the excess use of antibiotics is essential to combat the increase in antibiotic-resistant microorganisms

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[11, 12]. A reduction in antibiotic use results in fewer side effects, lower costs, and, in the long-term, leads to decreasing drug resistance. To limit antibiotic use, a rapid and accurate differentiation of clinically relevant bacterial LRTI from other, mostly viral causes is pivotal [13, 14].

The randomized ‘ProRESP’ intervention study recently assessed the ability of PCT measurements to identify bacterial LRTIs requiring antimicrobial treatment in the setting of an emergency department [15]. PCT was chosen as the test marker because of its advantages over CRP and other inflammatory markers, namely an earlier increase upon infection, a better negative predictive value, and the unattenuated increase in the presence of immunosuppressive medication (e.g., steroids in patients with COPD) [7]. PCT was measured using a rapid assay with a functional sensitivity of 0.06 µg/l (Kryptor® PCT, Brahms, Hennigsdorf, Germany). The assay time for PCT measurements was less than 20 minutes and results were routinely available within 1 hour (24 hours a day, 7 days per week).

Investigating physicians used an algorithm developed at University Hospital, Basel, Switzerland, to classify patients in the PCT group into four subgroups according to the probability of bacterial infection. The following PCT cut-off ranges were derived by calculating multilevel likelihood ratios and optimized for the setting of an emergency room and hospital:

) A PCT level of ‹ 0.1 µg/l suggested the absence of bacterial infection and the initiation or continuation of antibiotics was strongly discouraged. Antibiotic therapy could be considered in critically ill patients. If antibiotics were given, an early discontinuation of antibiotic therapy after 1 – 3 days was endorsed if PCT levels, checked daily, remained ‹ 0.1 µg/l.

) A PCT level between 0.1 and 0.25 µg/l indicated that bacterial infection was unlikely, and the initiation or continuation of antibiotics was discouraged. Anti- biotic therapy could be considered in high-risk patients. Again, if antibiotics were given, early termination was endorsed if PCT levels did not increase.

) A PCT level between 0.25 and 0.5 µg/l indicated a possible bacterial infection and the initiation or continuation of antibiotic therapy was encouraged.

) A PCT level of 8 0.5 µg/l strongly suggested the presence of bacterial infection and antibiotic treatment and continuation was strongly encouraged [15].

The same cut-offs were used regardless of whether or not patients had been pretrea- ted with antibiotics prior to admission to the emergency department. Re-evaluation of the clinical status and measurement of serum PCT levels was recommended after 6 – 24 hours in all persistently sick and hospitalized patients in whom antibiotics were withheld. The PCT algorithm could be overruled in patients with immediately life-threatening disease (e.g., patients with severe co-morbidity, emerging need for intensive care unit [ICU] admission during the initial follow-up, in patients with hemodynamic or respiratory instability, and in very ill patients with positive antigen test for legionellosis).

Physicians were advised that persistently elevated PCT levels may indicate a com- plicated course (e.g., empyema or abscess), while PCT levels may remain relatively low in localized infections. PCT levels were reassessed on days 4, 6 and 8 in hospitalized patients with ongoing antibiotic therapy, and in patients showing a worsening or delayed recovery of signs and symptoms, and antibiotics were discontinued using the PCT cut-offs defined above. In patients with very high PCT values on admission (i.e., 8 10 µg/l), discontinuation of antibiotic therapy was encouraged if levels decreased to below 80 – 90 % of the initial value. In patients with an initial PCT level

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8 10 µg/l and smaller reductions during follow-up, continuation of antibiotic treatment was encouraged.

For antibiotic stewardship in a medical ICU, modified cut-off ranges may be nec- essary. Since mean PCT levels are increased in a cohort of critically ill patients as compared to patients in an emergency room or hospital setting, the optimal thresh- olds of the cut-off ranges are likely to be higher. A suggested algorithm for a medical ICU based on an observational study [6] is shown in Figure 1.

In the ProResp study in the emergency room setting, the percentage of patients with LRTI who received antibiotic therapy in the PCT group was reduced by almost 50 %, compared with the standard group [15]. The clinical and laboratory outcome was similar in both groups. Reduced antibiotic use was most striking in acute bronchitis and acute exacerbations of COPD. COPD exacerbations offer a particular chal- lenge in terms of diagnosing an infectious cause. The majority of COPD patients have positive sputum culture results, although these do not necessarily imply an active infection. In the PCT group, positive culture rates were similar in patients in whom antibiotics were given or withheld, as were outcomes, underlining the limited diagnostic usefulness of sputum cultures in COPD. Since patients with COPD have an impaired pulmonary reserve and infection may be locally contained, a PCT cut- off level for withholding antibiotics of ‹ 0.1 µg/l is advisable in patients with severe disease. This was validated in the ProCOLD study, which included more than 200 patients [16].

In patients with community-acquired pneumonia (CAP), PCT levels are almost always high. Pneumonia is defined as inflammation of the pulmonary parenchyma,

Fig. 1. Procalcitonin-guided antibiotic stewardship.

Procalcitonin (PCT)-guided antibiotic therapy was successfully validated in more than 1200 patients with lower respiratory tract infections in the setting of the emergency room, primary care and hospital. For use in intensive care units, cut-offs have to be adapted to the local setting. Herein, cut-off ranges are proposed based on an observational study performed in the medical intensive care unit [6] and important aspects to be considered in any intensive care unit are highlighted in gray. The cut-off ranges may be even higher in a surgical intensive care unit or if applied in newborns where high PCT levels can be found even in the absence of an infection. Importantly, the course of PCT levels is more relevant than the initial value in critically ill patients. CAP: community-acquired pneumonia; PSI: pneumonia severity index

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which is often caused by a bacterial agent, mirrored in markedly elevated PCT levels. In CAP, antimicrobial therapy must be promptly initiated, because a delay in treatment is associated with increased mortality [17]. Thus, the primary value of PCT in CAP is not to reduce antibiotics, but to facilitate the differential diagnosis of new or progressing infiltrates. Accordingly, PCT-guidance could markedly lower the number of antibiotic courses in patients with infiltrates on chest X-ray unrelated to pneumonia.

Importantly, the optimal duration of antimicrobial therapy in CAP is largely unknown [18]. Most likely, it varies from patient to patient and is dependent, among other factors, on the severity of the disease, the adequacy of the host response, and the underlying microorganism. Current guidelines recommend antibiotic courses of 7 to 14 days, depending on illness severity and type of pathogen [19 – 21]. However, adherence to guidelines is variable [22] and physicians tend to treat longer, espe- cially in elderly patients with co-morbidities and in patients with severe CAP [15, 23]. Optimal duration of antibiotic therapy can be guided by clinical signs such as defervescence, decrease in sputum production and coughing, or improvement in general condition. However, the interpretation of the clinical response lacks stan- dardization and validation and is prone to interobserver variability [24].

The dynamics of PCT levels have prognostic implications, as persistently elevated levels are associated with adverse outcomes [25]. Conversely, decreasing PCT levels suggest a favorable outcome, usually showing a log-linear drop-off and a half life of 20 to 24 hours [26]. We assessed, in a randomized intervention trial, the “ProCAP”- study, the capability of PCT-guidance to shorten antibiotic duration in patients with all severity levels of CAP admitted to the emergency department. We demonstrated in more than 300 patients with CAP that PCT guidance allows the duration of antibiotic treatment to be safely reduced from a median of 12 to 5 days with a similar outcome after a follow-up of 6 weeks. Importantly, measures of clinical and laboratory outcome were similar in both groups [27].

Hormokines as Biomarkers for Prognostic Assessment in CAP

In patients with CAP, improved diagnostic assessment by PCT is important in the differentiation of infection from other, non-infectious infiltrates, and in guiding the duration of antibiotics. In addition, it is pivotal to predict the prognosis of CAP and to estimate CAP severity for guiding therapeutic options such as the need for hospital or intensive care admission, suitability for discharge, and choice and route of antimicrobial agents. The pneumonia severity index (PSI) is a widely accepted and validated severity scoring system that assesses the risk of mortality for pneumonia patients in a two-step algorithm [28]. However, it is complex, which jeopardizes its dissemination and implementation in everyday practice. Therefore, the CURB-65 score has been proposed as a simpler alternative. Additionally, various easy to deter- mine surrogate biomarkers have been proposed to predict disease severity in CAP patients, thus complementing the PSI score [29 – 31].

Another member of the CALC gene family is adrenomedullin, which is one of the most potent vasodilating agents and has additional immune modulating, metabolic properties [32 – 35]. Adrenomedullin also has bactericidal activity, which is further enhanced by modulation of complement activity and regulation. Thus, it is not sur- prising that serum adrenomedullin levels are elevated in sepsis [36]. The reliable measurement of adrenomedullin is challenging, since it is rapidly cleared from the

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circulation [32,33]. The more stable mid-regional fragment of pro-adrenomedullin (ProADM) directly reflects levels of the rapidly degraded active peptide adrenomedullin [37].

In our study in over 300 patients with CAP, proADM levels measured on admission emerged as good predictors of severity and outcome of CAP with a similar prognostic accuracy to the PSI and better prognostic accuracy than commonly measured clinical and laboratory parameters. Importantly, proADM levels could improve the prognostic accuracy of the PSI alone, acting as an additional margin of safety [38].

It is advisable to base the difficult task of prognostic assessment and treatment decisions on several and not only one, parameters, each mirroring different patho- physiological aspects. In this context, we also evaluated the prognostic value of atrial natriuretic peptide (ANP), a member of the family of natriuretic peptides. ANP reg- ulates a variety of physiological parameters including diuresis and natriuresis, and reduces systemic blood pressure. Mature ANP is cleaved from carboxyl-terminal amino acids of the prohormone of ANP. The N-terminal portion of the prohormone is secreted in the same molar ratio as ANP [39, 40]. Because of its longer half-life, the N-terminal portion of proANP, particularly the mid-region of this molecule (MR-proANP), has been shown to be a more reliable marker [40]. In CAP, the MR- proANP level may mirror both the inflammatory cytokine response correlated with the severity of pneumonia, and the presence of disease-relevant comorbidities, namely heart failure and renal dysfunction [41 – 44]. Therefore, we investigated MR- proANP levels in a well defined cohort of 545 consecutive patients with LRTI, to evaluate its prognostic use for severity of disease and outcome. Our study had two main findings. First, plasma MR-proANP levels were increased in LRTI with highest levels in CAP. On admission, MR-proANP levels were a better predictor of severity and outcome of CAP than commonly measured clinical and laboratory parameters and comparable to the PSI [45]. Thus, proADM and MR-proANP are helpful in the risk stratification of patients with CAP. Of course, biomarkers will always oversim- plify the interpretation of important variables and proADM and proANP are, therefore, meant to complement, rather than to replace, clinical judgment and/or validated severity scores.

Hormokines as Biomarkers for Prognostic Assessment in Sepsis

A reliable prognostic assessment is crucial, not only in CAP, but equally in sepsis.

Sepsis is the leading cause of death in critically ill patients in the United States. It develops in 750,000 people annually, and more than 210,000 of them die [46, 47].

Roughly 9 % of patients with sepsis progress to severe sepsis, and 3 % to septic shock [48]. Early and adequate diagnosis and risk assessment is pivotal for optimized care of critically ill patients. The APACHE II score was originally suggested as a prognostic scoring system in sepsis and not to be used for individual outcome prediction of sepsis patients [49]. However, despite its inherent limitations, outcome predictors are clearly helpful in identifying those septic patients with a high risk of death, who are more likely to benefit from treatment.

In an attempt to improve current sepsis definitions the use of readily measurable circulating biomarkers is recommended, in the PIRO concept, as an additional tool for the timely assessment and severity classification of septic patients and the prediction of mortality [50].

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We showed that proADM has a similar prognostic accuracy to the APACHE II score [51]. This prognostic usefulness is validated by our more recent data in patients with CAP [38]. ProADM may, therefore, prove to be an additional helpful tool for a broader prognostic classification of septic patients. Two main mechanisms may be responsible for the marked increase in circulating MR-proADM and mature adrenomedullin in sepsis. First, as a member of the CALC gene family, adrenomedullin is widely expressed and extensively synthesized during sepsis, similar to other calcitonin peptides, namely PCT and calcitonin-gene related peptides [26]. Bacterial endotoxins and pro-inflammatory cytokines upregulate ADM gene expression in many tissues both in vitro and in vivo in rodents and humans [52]. In addition, decreased clearance by the kidneys may be responsible in part for the increased levels in sepsis [36].

Hormokines as Mediators in Severe Infections

PCT is a potentially harmful mediator involved in the infection response. The administration of PCT to septic hamsters with peritonitis doubled their death rate.

Conversely, immunoneutralizing elevated PCT levels with a specific antiserum greatly increased survival in septic hamsters [53] and pigs [54], even when adminis- tered after the animals were moribund. Several characteristics of PCT favor its use as a therapeutic target. In contrast to the transiently increased classical cytokines, for which immunoneutralization trials in humans have been disappointing, the mas- sive increase in circulating PCT levels persists for several days. Furthermore, PCT is frequently increased in overt sepsis, its onset is early (within 3 hours), and the diagnostic accuracy of its measurement should greatly improve patient selection for any study of the therapeutic efficacy of PCT immunoneutralization and antibiotic therapy in humans.

Conclusion

Used in conjunction with optimal clinical assessment, hormokines can improve the diagnostic assessment of sepsis and its precursors (e.g., LRTI, including pneumonia) and can, thereby, guide and reduce antibiotic use. Importantly, hormokines can also improve the prognostic assessment of sepsis and pneumonia compared to other routinely used laboratory parameters or clinical assessment. The therapeutic promise of this approach needs to be explored further.

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