|Year : 2018 | Volume
| Issue : 3 | Page : 977-982
Evaluation of hepcidin as a biomarker for neonatal sepsis
Fadi M Elgendy1, Ahmed A Khatab1, Hassan S Badr1, Gehanabdel Fatah2, Ahmed M El Fishawy1
1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||08-Jan-2017|
|Date of Acceptance||19-Mar-2017|
|Date of Web Publication||31-Dec-2018|
Ahmed M El Fishawy
Sirs Ellyan, Menoufia Governate
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate serum hepcidin as a biomarker for neonatal sepsis.
Neonatal sepsis is one of the most important neonatal disorders frequently associated with high rate of mortality and morbidity. Hepcidin is the key iron regulator; the clear link between hepcidin and innate immunity may be used for detection of sepsis.
Patients and methods
This case–control study was carried out in the NICU of Menoufia University Hospital from April 2015 to October 2016. It was conducted on 60 neonates: 40 neonates admitted in NICU as they were suspected for neonatal sepsis based on clinical and hematological scores and 20 healthy outpatient neonates served as a control group. The newborn infants were both full term and preterm, and their ages ranged from 1 to 28 days. An informed consent was taken from the parents before their enrollment in the study. We evaluate the serum hepcidin level as a biomarker for neonatal sepsis; we compared the results of serum hepcidin regarding blood culture and C-reactive protein (CRP) in case and control groups. All participants were subjected to history taking, thoroughly clinical examination, and laboratory investigations (complete blood count, CRP, blood culture, and micro-erythrocyte sedimentation rate), and we measured the serum level of hepcidin in septic and control groups.
Serum levels of hepcidin were significantly higher in neonates with sepsis than in healthy neonates. Serum levels of hepcidin were significantly correlated with blood culture results and CRP levels. After antibiotic therapy, the serum level of hepcidin was significantly decreased as compared with its pretreatment level.
Hepcidin – the key iron regulatory hormone –is a promising acute-phase reactant that may be a useful adjunct test aiding in the evaluation of neonatal sepsis. Use of hepcidin includes in early-onset sepsis as well as late-onset sepsis, in both full-term and preterm neonates.
Keywords: biomarker, evaluation, hepcidin, neonate, sepsis
|How to cite this article:|
Elgendy FM, Khatab AA, Badr HS, Fatah G, El Fishawy AM. Evaluation of hepcidin as a biomarker for neonatal sepsis. Menoufia Med J 2018;31:977-82
|How to cite this URL:|
Elgendy FM, Khatab AA, Badr HS, Fatah G, El Fishawy AM. Evaluation of hepcidin as a biomarker for neonatal sepsis. Menoufia Med J [serial online] 2018 [cited 2020 Feb 28];31:977-82. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/977/248739
| Introduction|| |
Sepsis is a complex clinical syndrome that arises from the activation of an innate host response to danger. The septic response is an extremely complex chain of events involving inflammatory and anti-inflammatory processes, humoral and cellular reactions, and circulatory abnormalities. The diagnosis of sepsis and evaluation of its severity is complicated by the highly variable and nonspecific nature of the signs and symptoms of sepsis. The early diagnosis and evaluation of the severity of sepsis is very important for starting timely the specific treatment. Sepsis is a major cause of morbidity and mortality in neonates. The clinical signs are nonspecific. Blood culture is the gold standard for confirmation of diagnosis, but the results are available only after 48–72 h, and there are frequent false-negative culture results. Therefore, search for diagnostic markers of infection with high sensitivity and specificity is still required. Krause et al. described a 25-peptide molecule that was later called ‘hepcidin’ based on its hepatic expression and antimicrobial activity. Chromosome 19 contains the hepcidin antimicrobial peptide (HAMP) gene which codes for hepcidin, which is a key regulator of iron homeostasis that is synthesized mainly by liver and excreted in urine. Hepcidin secretion is increased by iron overload and inflammation, whereas it is decreased by hypoxia, anemia, increased erythropoiesis, and decreased iron stores. Hepcidin is not only an iron-regulatory hormone but also an important link between host defense and iron metabolism. It plays a dual function in innate immunity as it enhances intracellular sequestration of iron by degrading the membrane iron transporter ferroportin, thereby depriving pathogens of this essential mineral, and it has a direct antimicrobial activity. The clear link between the hepcidin molecule and innate immunity may be used for the detection of sepsis. During infection and inflammation, hepcidin synthesis is markedly increased by a mechanism that is independent of iron status or erythropoietic activity. The cytokine interleukin-6 (IL-6) is an important key inducer of hepcidin synthesis during acute inflammation. Other cytokines, including IL-22, activin B, and IL-1β, may also contribute. IL-6 and IL-22 can bind to its receptor to increase hepcidin expression through Stat3 (signal transducer and activator of transcription-3), but IL-1β and activin B increase HAMP transcription through the BMP-Smad signaling (bone morphogenetic protein-sons of mothers against decapentaplegic homolog). Endoplasmic reticulum stress turns on hepcidin transcription through cyclic AMP response element-binding protein H and/or C/EBP homologous protein. Lipopolysaccharides promote hepcidin activation in macrophages through toll-like receptor 4 signaling. Hepcidin has a critical role in inflammation and contributes to host defense by interfering with microorganism's access to iron. Iron is essential for nearly all microbes, and microbial pathogens use multiple and often complex uptake mechanisms to obtain iron. Disruption of these mechanisms attenuates microbial viability and pathogenicity. Infectious organisms may acquire the ability to both evade immune surveillance and resist antibiotics, but they cannot escape their basic iron requirement for growth. Hepcidin can divert iron away from pathogens, and the resulting inhibition of pathogen replication may allow time for immunity to develop and control infections before rapid microbial growth overwhelms the infected individual. The present study was performed to evaluate serum hepcidin as a biomarker for neonatal sepsis.
| Patients and Methods|| |
This case–control study was carried out in the NICU of Menoufia University Hospital from April 2015 to October 2016. It was conducted on 60 neonates: 40 neonates admitted in NICU as they were suspected for neonatal sepsis based on clinical and hematological scores and 20 healthy outpatient neonates served as a control group. The newborn infants were both full terms and preterm, and their ages ranged from 1 to 28 days. An informed consent was taken from the parents before their enrollment in the study.
All neonates admitted to NICU and suspected to have sepsis were eligible for the study.
Neonates with birth asphyxia (documented by arterial blood gases); neonates with congenital anomalies, inborn error of metabolism, congenital infections, and organ failure (e.g., renal failure); neonates with surgical problems or postoperative cases; and those with twin-twin transfusion syndrome or placenta abruption are excluded. Those with a history of blood transfusion or hemoglobin level less than 10 g/dl were also excluded because of the important role of hepcidin in anemia.
All the patients and controls were subjected to the following:
Full history taking, thoroughly clinical examination, and laboratory investigations [complete blood count (CBC), C-reactive protein (CRP), blood culture, micro-erythrocyte sedimentation rate (ESR)]. Moreover, we measured the serum level of hepcidin in septic and control groups. Case selection was based on clinical sepsis score more than 3 points according to Tollnar and hematological sepsis score more than 3 points according to Rodwell et al..
Laboratory investigations include the following:
- Routine laboratory studies
- CBC by ADVIA 2120 (Siemens Healthineers, Siemens Healthcare GmbH, Henkestr, Erlangen, Germany), an auto-counter supplied from SIEMENS
- Kidney function tests: blood urea and serum creatinine level using the open system autoanalyzer Synchron CX5 (Beckman Coulter (UK) Ltd, High Wycombe, United Kingdom)
- Liver enzymes: alanine aminotransferase and aspartate aminotransferase were measured on a Synchron CX-9autoanalyzer (Beckman Instruments Inc., Fullerton, California, USA)
- Quantitative measurement of the level of CRP using automatic autoanalyzer Integra 400 (Roche Diagnostics, Mannheim, Germany)
- Blood gases on AVL (Roche Diagnostics)
- Micro-ESR using capillary tubes fixed vertically against a premarked perpendicular line. The height of plasma column was measured after the first hour and reported as micro-ESR (mm/h)
- Blood cultures: it was done for 40 neonates with suspected sepsis under both aerobic and anaerobic condition. One milliliter of blood was collected under aseptic precautions, for the aerobic and anaerobic blood culture bottles, which was incubated at 37C° for 48 h. Subcultures were done every 48 h under both aerobic and anaerobic conditions. Identification of the growing colony was done using Gram-staining film and biochemical characters, and automatic microorganism identification was done by using of Vitek2 compact system (Vitek 2; BioMérieux Inc. Hazelwood, Missouri, USA)
- Serum hepcidin: it was done by capture enzyme-linked immunosorbent assay, and this detects the 25-amino acid mature form of hepcidin using a commercially available hepcidin kit (DRG International Inc., Springfield, NJ, USA)
Data were collected, tabulated, and statistically analyzed by statistical package for social sciences program software version 20 (SPSS Programming and Data management; SPSS Inc., Chicago, Illinois, USA).
Two types of statistics were done: descriptive and analytics.
- Descriptive statistics: quantitative data are expressed to measure the central tendency of data and diversion around the mean (x) and SD. Qualitative data were expressed in numbers and percentage.
- Analytic statistics included Student's t-test, χ2-test, and Mann–Whitney U-test
- Receiver operating characteristic (ROC) curve: the ability of a test to discriminate diseased cases from normal cases was evaluated using ROC curve analysis. ROC curves can also be used to compare the diagnostic performance of two or more laboratory or diagnostic tests
- Sensitivity: probability that a test result will be positive when the disease is present (true positive rate, expressed as a percentage)
- Specificity: probability that a test result will be negative when the disease is not present (true negative rate, expressed as a percentage)
- Positive predictive value (PPV): probability that the disease is present when the test is positive (expressed as a percentage)
- Negative predictive value (NPV): probability that the disease is not present when the test is negative (expressed as a percentage).
P value less than 0.05 was considered significant.
| Results|| |
The demographic data of the studied groups shows there is no significant statistical difference between patients and controls regarding age, sex, gestational age, mode of delivery, and Apgar score [Table 1].
Laboratory investigations of the studied groups show there is a significant statistical difference in CBC (hemoglobin, hematocrit value, white blood cells, total leukocytic count, immature leukocytes, immature to total leukocytes ratio, immature to mature leukocytes ratio, and platelet count), micro-ESR, CRP, serum albumin level, urea level, creatinine level, and aspartate aminotransferase. However, there is no significant difference in alanine aminotransferase, K, and Na levels [Table 2].
There is a significant statistical difference regarding serum hepcidin level between case and control groups, whereas there is a significant decrease in convalescent serum hepcidin level [Table 3].
There is no significant statistical difference between early-onset and late-onset sepsis regarding hepcidin, CRP hematological, and clinical scores of the septic group regarding time onset of sepsis [Table 4].
|Table 4: Hepcidin, C-reactive protein, hematological, and clinical scores of the septic group regarding sepsis onset|
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For hepcidin, CRP, hematological, and clinical scores of the septic group regarding gestational age, there is no significant statistical difference [Table 5].
|Table 5: Hepcidin, C-reactive protein, hematological, and clinical scores of the septic group regarding gestational age|
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ROC comparison between hepcidin, CRP, and micro-ESR shows hepcidin has a better accuracy in sepsis detection than CRP and micro-ESR [Table 6].
|Table 6: Receiver operating characteristic comparison between serum hepcidin, C-reactive protein, and micro-erythrocyte sedimentation rate|
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| Discussion|| |
The present study was performed to clarify the role of hepcidin in diagnosis of neonatal septicemia compared with other known measures such as blood culture and CRP. Our study was carried out on 60 newborn infants: 40 septicemic neonates (subjected to full clinical examination and laboratory investigations) and 20 neonates with no evidence of sepsis serving as a healthy control group. The members of case group have a mean gestational age of 36.2 weeks and a mean birth weight of 2.4 kg. They were divided into 19 preterm (47.5%) and 21 full term (52.5%), including 22 males (55%) and 18 females (45%). Moreover, the control group has a mean gestational age of 36.5 weeks and a mean birth weight of 2.6 kg and was divided into 10 preterm (50%) and 10 full-term (50%) infants, including 10 males (50%) and 10 females (50%).
In our study, there were no significant statistical differences between case and control groups regarding gestational age, weight, length, or head circumference. However, we found that the frequency of neonatal sepsis among male newborns was predominant, representing 55% of cases; similar result was observed in the study by Robinson et al.. Hyperstimulation Syndrome (HSS) was significantly higher in septic group than control group in our study, and we found that immature to total neutrophil ratio in the case group was significantly increased compared with the corresponding values in control group. These results correlated with those of Mondal et al.. Total leukocytic count was significantly higher in septic group than control group, and this comes in agreement with Rodrigo. Blood culture is a gold standard for the diagnosis of septicemia and should be performed in all cases of suspected sepsis before starting antibiotics. A positive blood culture with sensitivity of the isolated organism is the best guide to antimicrobial therapy. Blood cultures in our study were withdrawn as early as possible after admission in NICU, and the results showed that culture-proven sepsis was documented in 27 (67.5%) of 40 cases with suspected sepsis, including 16 cases (59%) owing to Gram-negative organisms, seven cases (26%) owing to Gram-positive organisms, and four cases (15%) owing to fungal infection. This is consistent with blood culture results of Dzwonek, who documented blood culture positive cases to be 69.2%. In the present study, blood culture results proved six cases (22.2%) infected by Klebsiella pneumonia, five cases (18.5%) by Escherichia coli, four cases (14.8%) by Candida albicans, three cases (11.1%) by Staphylococcus aureus, and three cases (11.1%) by coagulase-negative staphylococci, and these were the most common organisms, whereas the least common organisms were two cases infected by pseudomonas (7.4%), two cases by Enterococci (7.4%), one case by group B streptococcus (3.7%), and there was one case isolated from CSF culture infected by Gram-negative bacilli (3.7%). Our study revealed that E. coli was the most common pathogen in Eosinophil (EOS) (23.5%), whereas K. pneumonia was the most common pathogen in LOS% (21.7%). In our study, micro-ESR was significantly higher in the case group than control group. ROC curves for micro-ESR compared with blood culture shows area under the curve is more than 0.65, meaning the test is mildly accurate in diagnosis; sensitivity is 65%; and specificity is 55% at the cut-off point of 15 mm/h. This is similar to study done by Piccina et al.. Micro-ESR method is nonspecific and sedimentation is low in the newborn owing to high hematocrit level, limiting its use as an indicator of sepsis in neonates. In our study, we found that CRP levels were significantly higher in the case group than control group. The range of CRP was from 12 to 186 mg/dl, with mean 48.85 ± 31.02 mg/dl in septic group. The control group in our study had a negative CRP. Area under the curve for CRP is more than 0.82, meaning the test is moderately accurate in diagnosis; sensitivity is 67%; and specificity is 80% at the cut-off point of 12 mg/dl. This comes in agreement with the results of the study of Shyamala et al.. CRP is a component of the innate immune system, and increased levels are observed in response to severe bacterial infection as a classical acute-phase reactant; however, CRP elevation alone has insufficient specificity and limited sensitivity for diagnosis of neonatal infection.
Hepcidin is a type II acute-phase protein that provides a molecular link between inflammation, resulting anemia, and the regulation of iron metabolism. Hepcidin induction during infection causes depletion of extracellular iron, which is thought to be a general defense mechanism against many infections by withholding iron from invading pathogens. Recently, endogenous expression of hepcidin by macrophages and neutrophils in response to bacterial pathogens confirmed its role in innate immunity. The clear link between the hepcidin molecule and innate immunity may be used for the detection of sepsis. In our study, we found that serum level of hepcidin in the case group was significantly increased compared with the corresponding values in control group. The cut-off point that discriminates case from control group is 107.5 ng/ml. This comes in agreement with the results of the study by Motalib et al., who reported increase in serum hepcidin in early-onset neonatal sepsis, at the cut-off point of 105 ng/ml. In our study, the range of hepcidin in serum of term infants in the control group is 20–92 ng/ml and the mean ± SD is 51.9 ± 21.028 ng/ml, whereas the range of hepcidin in serum of preterm infants in the control group is 25–60 ng/ml and the mean ± SD is 40.4 ± 17.02 ng/ml. Cizemia et al. reported significant increase in hepcidin level of cord blood (>118.1 ng/ml) as well as significant increase in serum level of hepcidin in neonates with early-onset neonatal sepsis. However, Wu et al., reported increase in serum hepcidin in late- neonatal sepsis, at the cut-off point of 92.5 ng/ml, which correctly classified 91% of all infants (PPV 100%, NPV 87%, specificity 100%, and sensitivity 76%). When statistical analysis was restricted to infants with positive blood culture results, sensitivity of hepcidin was 90%, specificity 91%, NPV 97%, and PPV 75%. When statistical analysis of our study was restricted to infants with positive blood culture results, sensitivity of hepcidin was 92.5%, specificity 88%, NPV 93.5%, and the PPV 86% at the cut-off point of 172.5 ng/ml. In the current study, we found no significant correlation between hepcidin and gestational age, Apgar score, age, anthropometric measures, hemoglobin, or hematocrit value (%). Nevertheless, we found a positive correlation between the level of hepcidin and positive CRP. However, performance of hepcidin combined with CRP was not better than hepcidin alone. In our study, there was four-fold increase in the level of serum hepcidin between normal and septic neonates in both early-onset and late-onset sepsis, with no significant difference regarding sex, birth weight, or gestational age. The normal level of serum hepcidin was 46.15 ± 19.024 ng/ml in the control group, whereas the level of serum hepcidin in the case group was 196.05 ± 39.63 ng/ml. Our study revealed a significant association between positive blood culture results and serum levels of hepcidin; the mean serum hepcidin level in suspected cases of sepsis with negative blood culture result was 153.73 ± 22.38 ng/ml whereas in septic cases that were proved by positive blood culture result, the serum hepcidin level was 216.43 ± 28.25 ng/ml. In convalescent cases (within 5 days of therapy), the level of serum hepcidin was 137.84 ± 15.84 ng/ml. This indicates that there is a significant increase in serum hepcidin level in neonatal sepsis, whereas there is a significant decrease in serum hepcidin after treatment. So, we may suggest that hepcidin may be considered as a useful adjunct test aiding in the evaluation of neonatal sepsis.
| Conclusion|| |
Hepcidin – the key iron regulatory hormone – is a promising acute-phase reactant that may be a useful adjunct test aiding in the evaluation of neonatal sepsis. Use of hepcidin includes in early-onset sepsis as well as late-onset sepsis, in both full-term and preterm neonates.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]