|Year : 2016 | Volume
| Issue : 1 | Page : 20-24
Relative importance of inflammatory markers (erythrocyte sedimentation rate, C-reactive protein, procalcitonin, and interleukins-6) in neonatal sepsis
Satyaki Das, Jaydeb Ray
Department of Pediatrics, Institute of Child Health, Kolkata, West Bengal, India
|Date of Submission||17-Apr-2016|
|Date of Acceptance||04-May-2016|
|Date of Web Publication||2-Jun-2016|
AC-52/6, Salt Lake, Sector - 1, Kolkata - 700 064, West Bengal
Source of Support: None, Conflict of Interest: None
Objective: Early diagnosis and treatment decreases the mortality and morbidity of neonatal sepsis(NS). The aim of this study was to find out the level of different inflammatory markers in neonatal sepsis.
Methods: Forty two term neonates with non-sepsis (n = 17), clinical (n = 10) and proven (n = 15) sepsis were evaluated. Blood cultures were obtained and ESR, CRP, procalcitonin, IL-6 levels were measured. Statistical analysis was performed to look for association between NS and different inflammatory markers and to compare the strength of association among the markers.
Findings: Among the 42 neonates, CRP level was found to be elevated in 27 neonates, ESR in 22 neonates, both procalcitonoin and IL-6 in 24 neonates respectively. Both sensitivity and specificity of procalcitonin and IL-6 were high compare to CRP.
Conclusions: Blood culture significantly increases the proven sepsis status among neonates with suspected sepsis. ESR is a poor predictor of NS. Newer inflammatory markers namely procalcitonin and IL-6 were found to have greater value than CRP, but not ESR.
Keywords: Blood culture positivity, C-reactive protein, erythrocyte sedimentation rate, interleukin-6, neonatal sepsis, procalcitonin
|How to cite this article:|
Das S, Ray J. Relative importance of inflammatory markers (erythrocyte sedimentation rate, C-reactive protein, procalcitonin, and interleukins-6) in neonatal sepsis. BLDE Univ J Health Sci 2016;1:20-4
|How to cite this URL:|
Das S, Ray J. Relative importance of inflammatory markers (erythrocyte sedimentation rate, C-reactive protein, procalcitonin, and interleukins-6) in neonatal sepsis. BLDE Univ J Health Sci [serial online] 2016 [cited 2019 Mar 18];1:20-4. Available from: http://www.bldeujournalhs.in/text.asp?2016/1/1/20/183270
Neonatal sepsis (NS) has significant morbidity and mortality rates and it is still difficult to diagnose on presentation. For this reason, neonates with suspected sepsis are usually prone to empiric broad-spectrum systemic antibiotic therapy until sepsis can be excluded, and pathogens are identified in only a small proportion of those patients. This empirical overuse of antibiotics favors the development of antimicrobial-resistant organisms. , To prevent microbial resistance induced by unnecessary administration of antibiotics, a definite diagnosis should be made using laboratory tests with a higher diagnostic value. ,
The gold standard for diagnosis of bacterial sepsis is blood culture, although pathogens in blood cultures are only detected in approximately 62% of the cases. The diagnosis of sepsis is often based on clinical assessment, in combination with laboratory markers such as C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). 
Procalcitonin (PCT) is produced in the liver or by macrophages and is secreted as an acute-phase reactant into the blood during infection. PCT starts to rise 4 h after exposure to bacterial products and reaches peak concentration within 6-8 h. Recently, PCT has received considerable interest in the diagnosis of bacterial infections in both pediatric and adult populations, although its accuracy in NS diagnosis is still controversial. ,,] In a study by Vazzalwar et al.,  a PCT cutoff value of 0.5 ng/mL was found to be more sensitive than CRP in predicting late-onset NS in very low birth weight infants. One limitation of PCT as an early marker for early-onset NS is that its concentration continues to rise normally during the first 48 h after birth, with a peak concentration at 18-30 h. ,
Pro-inflammatory cytokines, especially interleukin-6 (IL-6) are primarily responsible for the initiation of effective defense against invasive pathogens.  IL-6 is produced by phagocytes and endothelial cells in response to infection and inflammation. Studies have reported that IL-6 cutoff values to diagnose sepsis have ranged from 18 to 31 pg/mL. , However, elevation of cytokines may occur normally after delivery, limiting their use as a diagnostic tool in the neonatal setting, especially in the immediate postpartum period. There are also several other variables, such as hypoxia, fetal distress, prematurity, antenatal steroids, and other meconium aspiration, that elevate cytokine levels and limit their use in diagnosing early-onset NS. 
This study aimed to evaluate the newer methods for the diagnosis of NS (determination of PCT and IL-6 levels) and compare them to the conventional sepsis marker CRP, ESR, and BACTEC blood culture. Because suspicion of NS is based on a number of known risk factors, clinical signs, and laboratory markers, identification of a sign or a marker that could predict the diagnosis of NS is of paramount importance.
Aims and objectives
1. To find out the level of different inflammatory markers in NS
2. To find out the correlation of different markers.
| Materials and Methods|| |
The present prospective observational study was conducted in term neonates (0-28 days) at the Institute of Child Health, Kolkata, from January 2015 to June 2015.
Newborns' clinical signs of NS included respiratory manifestations such as apnea (suspension of external breathing for at least 20 s), tachypnea (respiratory rate over 60 breaths/min), nasal flaring, retractions, cyanosis or respiratory distress and/or bradycardia (heart rate <80 beats/min in term neonates) and/or hypotonia, seizures, poor skin color, irritability, or lethargy. 
Neonates suspected to have congenital malformations and/or laboratory-confirmed TORCH infections were excluded from the study.
Study tools and methodology
After a thorough history and clinical examination, blood samples were aseptically obtained from each neonate within the 24 h of Neonatal Intensive Care Unit admission as follows: 0.5 mL was inoculated immediately into blood culture bottles for blood culture, 0.5 mL was collected in an ethylenediaminetetraacetic acid tube for ESR, and 1 mL was collected in plain tubes to separate serum, which was kept at −20°C until used for assessment of CRP, IL-6, and PCT. Blood culture bottles were directly incubated in BACTEC 9050 system (Becton Dickinson, New Jersey, USA). Positive cases were subjected to subculture and complete bacteriological identification according to the standard microbiological methods.  IL-6 measurement was performed using chemiluminescence immunoassay platform (ROCHE e420). PCT was assessed by sandwich immune assay (Piramal healthcare). 
Statistical analysis was performed using Student's t-test and Chi-square test to look for an association between NS and different inflammatory markers and also to compare the strength of association among the inflammatory markers, respectively.
| Results|| |
The studied neonates were divided into three groups: Group 1, proven NS; Group 2, clinical NS; and Group 3, negative infection status or nonsepsis [Bar diagram 1 [Additional file 1]].
Group 1 included neonates with positive blood culture. Group 2 included neonates with negative blood culture who had positive clinical signs consistent of sepsis and positive sepsis screen based on band cells >20%, polymorphocytosis, elevated CRP level, and other parameters according to Malik et al. Group 3 included neonates suspected of having sepsis who had negative blood culture and negative sepsis screen [Table 1].
Blood cultures were positive in 15 cases; the most common isolated organisms were Gram-negative bacilli (Klebsiella, Escherichia coli, Enterobacter, Serratia marcescens, and Acinetobacter) and Gram-positive organisms (Streptococcus agalactiae, Group B Streptococci [GBS], and coagulase-negative staphylococci [CoNS]) [Table 3]. The CRP was significantly elevated (cutoff value = 5 mg/L)  in 13 patients in Group 1, 9 patients in Group 2, and 5 patients in Group 3. ESR was significantly elevated (cutoff value >20 mm in male and 15 mm in female at 1 st h)  in 8 patients in Group 1, 5 patients in Group 2, and 10 patients in Group 3. IL-6 was significantly increased (cutoff value = 30 pg/mL)  in 24 cases (12 in Group 1, 7 in Group 2, and 5 in Group 3). PCT was found to be elevated (cutoff value = 1 ng/mL)  in 24 cases: 13 cases in Group 1, 8 in Group 2, and only 5 cases in Group 3 [Table 2]. Sensitivity and specificity of different methods are shown in [Table 4] [Bar diagram [Additional file 2]].
|Table 2: Comparison between results of C-reactive protein, erythrocyte sedimentation rate, interleukin-6, and procalcitonin among the studied groups|
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|Table 3: Results of polymerase chain reaction, hs - C - reactive protein, interleukin - 6, and procalcitonin in relation to the isolated organisms from blood of the studied groups [Bar diagram 2]|
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|Table 4: Sensitivity, specificity, and predictive values of the studied tests|
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| Discussion|| |
NS is still one of the major causes of morbidity and mortality among newborns in developing countries. It is considered a life-threatening clinical emergency that necessitates urgent diagnosis and treatment. ,, The causative organisms of NS vary in developed and developing countries. In this study, Gram-negative organisms such as Klebsiella (five isolates) and E. coli (four isolates) were the most common causative organisms followed by CoNS (two isolates). Newborns are most probably infected by Gram-negative organisms from the vaginal and fecal flora of the mother and the environment during delivery. On the other hand, other investigators found that CoNS was the most common causative organism of NS.  However, the mortality rate due to CoNS was lower than that due to Gram-negative bacilli. Similarly, Simonsen et al. reported that while GBS was the most common etiologic agent of NS, E. coli was the most common cause of mortality.
Since sepsis is a systemic inflammatory response to infection, blood culture is still considered the gold standard method for the diagnosis of NS.  In this study, only 15 out of 42 neonates who were suspected of having NS had positive blood culture. This low sensitivity (35.71%) may be attributed to inoculation of only 0.5 mL of blood or to the fact that about 60%-70% of infants had a low level of bacteremia. For optimal results, blood culture requires about 6 mL of blood, which is not feasible. ,
Determination of CRP is one of the most commonly used laboratory tests for the diagnosis of NS. It is also useful for monitoring the response to treatment and guiding antibiotic therapy. , ESR measures the distance (mm) that red blood cell has fallen after 1 h in a vertical column under the influence of gravity. The ESR measures the rate at which the red blood cells get separated from the plasma and fall to the bottom of a test tube. The rate is measured in mm/h.
PCT is an acute-phase reactant produced both by hepatocytes and macrophages. It begins to rise 4 h after exposure to bacterial infection; the PCT response is more rapid than that of CRP. Therefore, it is an attractive alternative for the detection of NS because PCT levels remain high compared with CRP. PCT is also useful in predicting the severity of infection, response to treatment, and outcome.  In contrast to CRP, infants with other clinical conditions such as trauma, meconium aspiration, and hypoxemia have normal or minimal elevation in PCT. , IL-6 is a pro-inflammatory cytokine produced by monocytes and macrophages activated by bacterial infection. IL-6 can be detected in blood earlier than CRP during the course of NS. Like most cytokines, IL-6 does not cross the placental barrier; therefore, its increased level may predict the possibility of NS during the first few hours of life. 
In our study, CRP level was found to be elevated in 27 neonates, with 88% sensitivity and 70.59% specificity. ESR level was found to be elevated in 22 neonates with 52% sensitivity and 41.18% specificity. In this study, it was found that PCT level was significantly elevated in 24 neonates suspected to have NS, with sensitivity of 84% and specificity of 70.59% [Bar diagram 3 [Additional file 3]]. In other studies, similar results were demonstrated where PCT sensitivity varied between 83% and 100% and specificity varied between 70% and 100%.  Our results support the findings of others  who suggested that PCT can be more accurate than CRP for the diagnosis of NS. IL-6 was found to be raised in 24 neonates, with 76% sensitivity, 70.59% specificity, and positive likelihood ratio of 2.58. In addition, IL-6 was mostly positive within 24 h after the onset of NS. Similar to our results, IL-6 was reported to have high sensitivity (76.9%), specificity (73.68%), positive predictive value (80%), and negative predictive value (70%). 
The limitations of our study were small sample size, technical difficulties in sample collection, and delayed reporting of organism detected by blood culture. In addition, it is arguable that CoNS detected by blood culture is really attributed to sepsis or merely a contamination.
| Conclusions|| |
Blood culture significantly increased the proved sepsis status among neonates suspected to have NS. None of the studied markers (CRP, ESR, PCT, and IL-6) could be used individually to confirm or exclude the diagnosis of NS, and combination with other hematological markers and clinical observation is an essential issue. ESR is a poor predictor of NS. Newer inflammatory markers, namely PCT and IL-6 were found to have greater value than ESR, but not CRP; however, further studies are needed to confirm these findings.
The authors wish to thank the Director Dr. Apurba Ghosh for giving us permission to publish this article and also to Surupa Basu, Assistant Professor, Department of Biochemistry and Dr. Suman Poddar, Department of Microbiology, for proving the data related to this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Shah BA, Padbury JF. Neonatal sepsis: An old problem with new insights. Virulence 2014;5:170-8.
Reier-Nilsen T, Farstad T, Nakstad B, Lauvrak V, Steinbakk M. Comparison of broad range 16S rDNA PCR and conventional blood culture for diagnosis of sepsis in the newborn: A case control study. BMC Pediatr 2009;9:5.
Abdollahi A, Shoar S, Nayyeri F, Shariat M. Diagnostic value of simultaneous measurement of procalcitonin, interleukin-6 and hs-CRP in prediction of early-onset neonatal sepsis. Mediterr J Hematol Infect Dis 2012;4:e2012028.
Shahian M, Pishva N, Kalani M. Bacterial etiology and antibiotic sensitivity patterns of early-late onset neonatal sepsis among newborns in Shiraz, Iran 2004-2007. IJMS 2010;3:293-8.
Benitz WE. Adjunct laboratory tests in the diagnosis of early-onset neonatal sepsis. Clin Perinatol 2010;37:421-38.
López Sastre JB, Pérez Solís D, Roqués Serradilla V, Fernández Colomer B, Coto Cotallo GD, Krauel Vidal X, et al.
Procalcitonin is not sufficiently reliable to be the sole marker of neonatal sepsis of nosocomial origin. BMC Pediatr 2006;6:16.
Døllner H, Vatten L, Austgulen R. Early diagnostic markers for neonatal sepsis: Comparing C-reactive protein, interleukin-6, soluble tumour necrosis factor receptors and soluble adhesion molecules. J Clin Epidemiol 2001;54:1251-7.
Vazzalwar R, Pina-Rodrigues E, Puppala BL, Angst DB, Schweig L. Procalcitonin as a screening test for late-onset sepsis in preterm very low birth weight infants. J Perinatol 2005;25:397-402.
Arnon S, Litmanovitz I. Diagnostic tests in neonatal sepsis. Curr Opin Infect Dis 2008;21:223-7.
van Rossum AM, Wulkan RW, Oudesluys-Murphy AM. Procalcitonin as an early marker of infection in neonates and children. Lancet Infect Dis 2004;4:620-30.
Ng PC. Diagnostic markers of infection in neonates. Arch Dis Child Fetal Neonatal Ed 2004;89:F229-35.
de Bont ES, Martens A, van Raan J, Samson G, Fetter WP, Okken A, et al.
Diagnostic value of plasma levels of tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) in newborns with sepsis. Acta Paediatr 1994;83:696-9.
Turner D, Hammerman C, Rudensky B, Schlesinger Y, Schimmel MS. The role of procalcitonin as a predictor of nosocomial sepsis in preterm infants. Acta Paediatr 2006;95:1571-6.
Malik A, Hui CP, Pennie RA, Kirpalani H. Beyond the complete blood cell count and C-reactive protein: A systematic review of modern diagnostic tests for neonatal sepsis. Arch Pediatr Adolesc Med 2003;157:511-6.
Camacho-Gonzalez A, Spearman PW, Stoll BJ. Neonatal infectious diseases: Evaluation of neonatal sepsis. Pediatr Clin North Am 2013;60:367-89.
Stocker M, Hop WC, Van Rossum AM. Neonatal procalcitonin intervention study: Effect of procalcitonin-guided decision making on duration of antibiotic therapy in suspected neonatal early-onset sepsis: A multi-centre randomized superiority and non-inferiority intervention study. BMC Pediatr 2010;10:89-97.
Laforgia N, Coppola B, Carbone R, Grassi A, Mautone A, Iolascon A. Rapid detection of neonatal sepsis using polymerase chain reaction. Acta Paediatr 1997;86:1097-9.
Ozkan H, Cetinkaya M, Koksal N, Celebi S, Hacimustafaoglu M. Culture-proven neonatal sepsis in preterm infants in a neonatal intensive care unit over a 7 year period: Coagulase-negative Staphylococcus as the predominant pathogen. Pediatr Int 2014;56:60-6.
Simonsen KA, Anderson-Berry AL, Delair SF, Davies HD. Early-onset neonatal sepsis. Clin Microbiol Rev 2014;27:21-47.
Goldstein B, Giroir B, Randolph A; International Consensus Conference on Pediatric Sepsis. International pediatric sepsis consensus conference: Definitions for sepsis and organ dysfunction in pediatrics. Pediatr Crit Care Med 2005;6:2-8.
Hornik CP, Benjamin DK, Becker KC, Benjamin DK Jr., Li J, Clark RH, et al.
Use of the complete blood cell count in early-onset neonatal sepsis. Pediatr Infect Dis J 2012;31:799-802.
Kellogg JA, Ferrentino FL, Goodstein MH, Liss J, Shapiro SL, Bankert DA. Frequency of low level bacteremia in infants from birth to two months of age. Pediatr Infect Dis J 1997;16:381-5.
Bender L, Thaarup J, Varming K, Krarup H, Ellermann-Eriksen S, Ebbesen F. Early and late markers for the detection of early-onset neonatal sepsis. Dan Med Bull 2008;55:219-23.
Hofer N, Zacharias E, Müller W, Resch B. An update on the use of C-reactive protein in early-onset neonatal sepsis: Current insights and new tasks. Neonatology 2012;102:25-36.
Chiesa C, Panero A, Rossi N, Stegagno M, De Giusti M, Osborn JF, et al.
Reliability of procalcitonin concentrations for the diagnosis of sepsis in critically ill neonates. Clin Infect Dis 1998;26:664-72.
Zeitoun AA, Gad SS, Attia FM, Abu Maziad AS, Bell EF. Evaluation of neutrophilic CD64, interleukin 10 and procalcitonin as diagnostic markers of early- and late-onset neonatal sepsis. Scand J Infect Dis 2010;42:299-305.
Whicher J, Bienvenu J, Monneret G. Procalcitonin as an acute phase marker. Ann Clin Biochem 2001;38:483-93.
Mehr S, Doyle LW. Cytokines as markers of bacterial sepsis in newborn infants: A review. Pediatr Infect Dis J 2000;19:879-87.
López Sastre JB, Solís DP, Serradilla VR, Colomer BF, Coto Cotallom GD, de Hospitales Castrillo G. BMC Pediatr 2007;7:9. DOI: 10.1186/1471-2431-7-9.
Adib M, Bakhshiani Z, Navaei F, Fosoul FS, Fouladi S, Kazemzadeh H. Iran J Basic Med Sci 2012;1:777-82.
Noor MK, Shahidullah M, Mutanabbi M, Barua C, Mannan MA, Afroza S. Comparison between CRP and IL-6 as early markers of neonatal sepsis. Mymensingh Med J 2008;17:72-6.
[Table 1], [Table 2], [Table 3], [Table 4]