Proadrenomedullin as a prognostic biomarker for critically-ill septic children

Background: The use of new biomarkers is a promising tool to assess risk of mortality among septic children. The aim of the study was to conrm prognostic and diagnostic value of pro-ADM as a marker in critically ill septic children. Methods: This study was conducted on 40 patients who were admitted at pediatric intensive care unit (PICU), diagnosed as sepsis in addition to 40 patients enrolled as control group. Serum pro-ADM level was measured for both cases and controls. Results: The level of pro-ADM was signicantly higher in cases than controls (p-value <0.001). However, no correlation between level of pro-ADM and outcome (p value 0.720) was found. Conclusion: The pro-ADM level was higher in cases than control, thus proved its diagnostic value but can’t be used as prognostic factor. Future studies are recommended and daily measurements are warranted to study its prognostic value.


Methods:
This was a prospective observational study conducted on patients with sepsis admitted to pediatric intensive care units (PICUs) in Cairo University Children Hospital during 6 months interval from January 2016 to July 2016.
A total of 80 participants were included in the study; Group 1: 40 patients (1 month to 12 years) diagnosed with sepsis and requiring PICU stay and Group 2: 40 age and gender-matched healthy children as a control group. The diagnosis of sepsis was con rmed according to the International guidelines of sepsis (8).
Children with immediate postoperative cardiac surgery, chronic lung disease and immunode ciency were excluded from the study. These pathologies were excluded because they may affect circulating ADM.
Full history taking (including age, sex and original diagnosis) and Clinical examination for all patients was recorded including; vital signs [heart rate, respiratory rate, temperature (low grade fever is de ned as temperature < 38.5 and high grade fever as temperature > 38. 5) and blood pressure], capillary re ll time [considered normal up to 2 sec], pupillary reaction, urine output over 24 hours [normal range 1 to 3 ml/kg/hour] and Glascow coma scale.
Grading of sepsis was reported according to international guidelines of sepsis into infection, systemic in ammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock and multi-organ dysfunction syndrome (MODS) (8).
Pro-ADM serum level was done by Enzyme-linked immune-sorbent assay (ELISA).
Principles of the assay: The kit assay Human Pro-ADM level in the sample, Puri ed Human Pro -ADM antibody was used to coat microtiter plate wells, solid phase antibody was made, then pro-ADM was added to wells, combined pro-ADM antibody which with horseradish peroxidase (HRP) labeled, became antibody-antigen-enzyme-antibody complex. After washing completely, tetramethybenzidine substrate (TMB) solution was added.
TMB substrate became blue color at HRP enzyme-catalyzed. Reaction was terminated by the addition of a sulphuric acid solution and the color change was measured spectrophometically at a wavelength of 450 nm. The concentration of pro-ADM in the samples was then determined by comparing the optical density of the samples to the standard curve.

Statistical Analysis:
Data was analyzed by SPSS statistical package version 17. Excel computer program was used to tabulate the results, and represent it graphically. Quantitative variables were expressed as mean and standard error. Qualitative variables were expressed as count and percent.
One Way ANOVA was used to declare the signi cant difference between groups at p < 0.05. Duncan multiple comparison test at p < 0.05 was used to declare the signi cant between each two groups. Chi square test used to declare the signi cant difference in the distribution between groups at p < 0.05 (11).

Results:
48 children (60%) were males and 32 (40%) were females. The mean age of cases was 14.63 ± 2.60 months while the mean age of controls was 16.22 ± 2.84, with no statistically signi cant difference between the 2 groups (p = 0.718).
Level of pro-ADM showed a statistically signi cant difference between cases (mean 46.47 ± 9.87) and controls (mean 5.73 ± 0.20) (p < 0.001). Sixteen cases (40%) died during the study while 24 cases (60%) were discharged. Correlations between the level of pro-ADM and age and laboratory investigations in the case group revealed a positive correlation between pro-ADM and CRP level. Table (3) shows correlation between outcome of cases and PRISM score, CRP, inotropic score and pro-ADM level. There were signi cant positive correlations between outcome and both PRISM and inotropic scores.
Table (4) shows correlation between different grades of sepsis and CRP. It shows statistically signi cant correlation between different grades of sepsis and CRP.

Discussion:
Sepsis is a systemic in ammatory reaction triggered by an infective agent (12). Severe sepsis may result in systemic in ammation, multi-organ failure and septic shock. It is one of the major health problems all over the world and a common reason for intensive care unit (ICU) admission (13).
Infections and sepsis are accompanied by clinical signs such as changes in body temperature and tachycardia which are not sensitive or speci c for sepsis. They have only moderate sensitivity and speci city and are not early markers due to the time taken to produce a reaction (14). General in ammatory markers such as leukocytosis and blood cultures together with CRP and procalcitonin (PCT) are commonly used to complement the diagnosis. ADM has been proposed as a useful biomarker for evaluating disease severity and risk of death (15).
Pro-ADM correlates well with other markers such as IL-6 (interleukin 6) and CRP as a predictor of prognosis in sepsis. In adults, elevations of pro-ADM were found in different stages of septic patients as systemic in ammatory response syndrome (SIRS), sepsis, and septic shock (4).
On the contrary, Christ-Crain et al., 2006 (16) found that 53/101 patients (52.5%) had sepsis, severe sepsis or septic shock, while 48 patients (47.5%) had systemic in ammatory response syndrome. This means that our patients started coming to the hospital after reaching late stage of disease evolution.
Twenty four cases were males (60%) and 16 were females (40%) with no signi cant correlation between sex and level of proadrenomedullin.
Similarly Jordan et al., 2014 (17) found that 59/95 patients involved in their study were males (62%) and the rest were females. This revealed that incidence of sepsis is more common in males than females. This may be due to sex-related hormonal secretion, different patterns of pro-in ammatory and antiin ammatory mediators in response to severe sepsis and more favorable hormonal and immunologic pro le in females (18).
In our study, level of proadrenomedullin was signi cantly higher in cases than controls with p-value < 0.001. This is in agreement with Oncel et al., 2012 (4), who revealed signi cantly higher levels of pro-ADM in cases with clinical and proven sepsis compared to healthy controls.
Also Christ-Crain et al., 2006 (16), measured pro-ADM in patients with different grades of sepsis and found that it was signi cantly higher in all sepsis groups compared to healthy controls or non-infected critically-ill patients.
A physiological explanation for the increase of circulating MR-pro-ADM could be because being a member of the calcitonin gene family, it is expressed and highly synthesized during sepsis similar to other calcitonin peptides such as PCT (19). In addition, bacterial endotoxins and proin ammatory cytokines may contribute to the upregulation of ADM gene expression in different tissues (20).
In order to prove the prognostic value of proadrenomedullin we correlated between its level, grades of sepsis (p-value 0.790), PRISM III score (p-value 0.6), outcome of patients either discharged or died (pvalue 0.720) and inotropic score (p-value 0.616) but no correlation was found. Also Guignant et al., 2009 (21) who performed their study on septic adult patients, revealed that MR-pro-ADM levels were signi cantly higher in non survivors.
In our study, correlation between CRP and grades of sepsis revealed positive correlation as when increasing grade of sepsis, CRP increases (p-value 0.005) which proves better prognostic value than pro-ADM while by correlation between CRP and outcome of patients we did not nd any correlation between them(p-value 0.262) similar to pro-ADM.
Against our study, Christ-Crain et al., 2006 (16) concluded the superior prognostic accuracy of MR-pro-ADM over other biomarkers such as CRP and PCT as the mean plasma MR-pro-ADM levels were signi cantly higher in non survivors. Similar results were obtained by Hagag et al., 2011 (22), who found that pro-ADM was lower in survivors in contrast to non-survivor cases; on the other hand CRP was not signi cantly different between survivors and non-survivors. Wang and Kang, 2010 (23), clari ed that, on the rst day of ICU admission, pre-atrial natriuretic peptide (pre-ANP) and pro-ADM in patients with sepsis, severe sepsis, or septic shock increased in non-survivors as compared with survivors, while other markers of infection and in ammation (CRP, IL-6 and PCT) were not which shows that pre-ANP and pro-ADM are better in predicting the severity of septic patients.
In our study, also by correlating between outcome and PRISM III, a signi cant correlation (p value 0.023) was found, as the higher the PRISM score, the incidence of mortality increases. Also a signi cant correlation was found between outcome and inotropic score (p-value 0.007), as the higher the inotropic score, the incidence of mortality increases. This showed that clinical evaluation by PRISM score and inotropic score can predict the outcome of patients. Jordan et al., 2014 (17), suggested the combined use of PRISM III and ADM as predictors of mortality in pediatric patients.
Our study showed positive correlation between pro-ADM and CRP (p-value 0.01). This justi es the usage of second parameters for diagnosis as combination between pro-ADM and other biomarkers as CRP.
Oncel et al., 2012 (4) found that pro-ADM levels decrease rapidly compared to CRP suggesting that instead of being used alone, combination with conventional acute-phase reactants may be more useful to diagnose and follow-up patients with sepsis.

Conclusion:
Pro-ADM is a good diagnostic biomarker for sepsis being higher in cases than controls. It cannot be used as prognostic factor as no correlation between its level and outcome. Clinical assessment of sepsis by PRISM III and inotropic scores have better prognostic assessment for prediction of outcome of cases.

Supplementary Files
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