The systemic changes associated with inflammation especially with infections, are collectively called the Systemic Inflammatory Response Syndrome (SIRS) or the acute phase response. These changes are reactions to cytokines whose production is stimulated by bacterial products, toxins and other inflammatory stimuli. The acute phase response consists of several clinical manifestations including fever and pathological markers such as changes in cytokines and leucocytes, ESR, C Reactive Protein (CRP), Procalcitonin etc. Information regarding these markers of infection and appropriate interpretation of their laboratory estimations is quite useful to the clinicians in their daily practice.1,2 When clinical picture is supported by markers of infection, appropriate culture specimens are obtained and antibiotic therapy is started. The antibiotic regimen may be modified subsequently on the basis of culture reports and response of the patient to the treatment.2
Leucocyte Abnormalities :
The total leucocyte count has a low predictive value for diagnosis of infection because of wide range of normal counts from 8,000 to 20,000 / cu.mm. Leucocytosis is a common feature of inflammatory reactions, especially those induced by bacterial infections. The leucocyte count usually increases to 15,000 to 20,000 cells/cu.mm.1,2 Occasionally, it may reach to extraordinarily high levels of 40,000 to 1,00,000 cells/cu.mm, referred to as leukemoid reactions. This is due to accelerated release of cells from the bone marrow postmitotic reserve pool caused by cytokines including interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF). Leukemoid reaction is commonly associated with B.pertussis and tuberculous infections.1,2 Prolonged infection also induces proliferation of precursors in the bone marrow, caused by increased production of Colony Stimulating Factors (CSFs). Thus, the bone marrow output of leucocytes is increased to compensate for the loss of these cells in the inflammatory reaction.3 Neonatal septicemia is usually associated with leucopenia (< 5000/cu.mm) or absolute neutropenia (<1000/cu.mm).4,5,6 A band neutrophil is an immature neutrophil, wherein the width of the narrowest segment of the nucleus is more than one third of the broadest segment. The band cell count of more than 20 percent and band count to total neutrophil count ratio of equal to or more than 0.2 is considered as 80 to 90 percent sensitive test for diagnosis of neonatal septicemia.3,4,5,6 In addition to it, toxic granulation and vacuolization are other morphological changes seen in neutrophils with septicemia.
Viral infections such as infections mononucleosis, mumps and rubella produce a leucocytosis by virtue of an absolute increase in the number of lymphocytes (lymphocytosis).1,2 Hay fever and parasitic infestations are known for eosinophilia. Enteric fever is usually associated with low leucocyte count and eosinopenia.1 Thrombocytopenia can occur during the initial phase of sepsis because of platelet consumption or vascular sequestration, whereas reactive thrombocytosis can be seen later as a result of bone marrow stimulation by cytokines. Falciparum malaria, enteric fever, dengue haemorrhagic fever and other infections are known for their association with thrombocytopenia and leucopenia (Table-I). In enteric fever platelets usually fall in the second week of illness, whereas in others they fall within the first week itself. Drop of haemoglobin is the characteristic of malaria, while rising hematocrit indicates Dengue fever.
Malaria (especially falciparum malaria), acute viral and bacterial infections are known to cause anaemia acutely.2 Non specific viral infections can cause coomb's positive hemolytic anemia. Mycoplasma pneumoniae may present with haemolytic anaemia with elevated titres of cold haemagglutinating antibodies. Transient erythroblastopenia is an acute phase response present during many infections. Hemophagocytic syndromes leading to pancytopenia can follow many infections especially due to herpes viruses, parvovirus B19, salmonella etc. Chronic infections like tuberculosis, urinary tract infection, bacterial endocarditis and chronic osteomyelitis are known to cause anaemia.1,2,3

Table-I Interpretation of CBC


Erythrocyte Sedimentation Rate (ESR) :
Acute phase proteins are plasma proteins, mostly synthesized in the liver, whose plasma concentrations may increase several hundred fold as part of the response to inflammatory stimuli. Three of the best known examples of these proteins are C-reactive protein, fibrinogen and serum amyloid A protein. The rise in fibrinogen causes erythrocytes to states (rouleaux) that sediment more rapidly at unit gravity than do individual erythrocytes. This is the basis for measuring the erythrocyte sedimentation rate (ESR).1,3 ESR is a simple test for the systemic inflammatory response, caused by number of stimuli incl. LPS.
The ESR is elevated in most bacterial and mycobacterial infections and is normal or mildly elevated in uncomplicated viral infections. Elevated ESR has poor discriminating power as a single test to predict bacterial infection in children with non specific, febrile illness of shorter duration. ESR is expected to be abnormal if a significant tissue focus of bacterial infection exists. ESR more than 50 mm/hour provides impetus for more extensive evaluation that can reveal localized bacterial infection, endocarditis, disseminated tuberculous infection or certain fungal infections. Extreme elevation of ESR (more than 100 mm/hour) is characteristic of certain conditions such as infective endocarditis, miliary tuberculosis, Kawasaki disease, sarcoidosis, malignancy, collagen disorders etc.1,2
In children with infectious diseases, abnormally low ESR is most frequently a sign of disseminated intravascular coagulopathy, reflecting low plasma fibrinogen concentration. Abnormally shaped RBCs (sickle cell, spherocytosis) and polycythemia prevent compact rouleaux formation and lower ESR. High dose of steroids and salicylates have been reported to lower ESR. 1,2,3
Micro-ESR is a simple marker for neonatal infection. It is not a very reliable marker. Its normal value is 6 mm during the first 3 days of life. By the end of first month, maximum fall may be upto 11 mm. During the neonatal period value of micro ESR more than 15 mm is considered suggesting infection. Micro ESR is obtained by collecting capillary blood in a standard pre-heparinized micro-hematocrit tube with 75 mm length, internal diameter of 1.1 mm and outer diameter 1.5 mm.4,5,6

Acute Phase Proteins :
Most of the acute phase proteins increase in plasma following inflammatory stimuli. There are two acute phase proteins namely prealbumin and transferrin called negative reactants as they decrease with infection and returns to normal with recovery.4 (Table-II).
Table-II Acute Phase Proteins :
Those increasing with inflammation.
• C-Reactive Protein
• Procalcitonin
• Cytokines (IL-6 and IL-Ira)
• Alpha-1-acid glycoproteins
• Heptoglobin (Alpha-2 glycoprotein)
• Alpha-1-antitrypsin
• Fibrinogen
Those decreasing with inflammation.
• Prealbumin
• Transferrin

Cytokines :
The important limit of hematological indices for early diagnosis of sepsis is the time required for the test to become positive. It takes several hours for leucocyte indices and acute phase reactants to change significantly after the onset of reaction.7 (Fig.1)
The cascade of events initiated by the bacterial infection usually begins with the activation of macrophages and release of inflammatory cytokines and growth factors. So increased plasma levels of cytokines is the primary host response to an inflammatory insult. Cytokines are glycoproteins released by macrophages, monocytes, lymphocytes and endothelial cells.1,7 Tumor necrosis factor. Alpha (TNF-a), interleukin-Beta (IL-b) and Interleukin-6 (IL-6) are major inflammatory cytokines while interleukin-3 (IL-3) and Colony Stimulating Factors (CSFs) are important growth factors. Figure-1 shows kinetics of various markers of the infection following endotoxin challenge in human volunteers.

Fig.1 : Kinetics of various markers of the inflammatory host response after endotoxin challenge in human volunteers. CRP, C-reactive protein; IL, interleukin; PCT, procalcitonin; TNF, tumor necrosis factor.

Tumor Necrosis Factor – Alpha (TNF-a):
It is considered the likely initiating factor in the activation of host response and subsequent cytokine release during infection with concentration increasing to 24 times (828 ng/L) to their preinfection concentration. Difficulties in using TNF-a for diagnosis of sepsis arise from its central role in the inflammatory response, short term concentration in response to bacterial infection and its short half life.7

Interleukin-6 (IL-6) :
IL-6 is a pleiotropic cytokine involved in many aspects of the immune system. It is synthesized by a number of cells such as monocytes, endothelial cells and fibroblasts after TNF and IL-1 stimulation. IL-6 is the major inducer of hepatic acute phase protein synthesis including CRP and fibrinogen. In most cases of neonatal sepsis, IL-6 increases rapidly, several hours before the increase in the concentration of CRP and decreases within 24 hours to undetectable levels. The short half life of IL-6 is caused by binding to plasma proteins, early clearance in the liver and inhibition by other cytokines. When used as a marker of infection, IL-6 has good sensitivity and specificity.8,9
A recent study showed that IL-6 and interleukin-1 receptor antagonist (IL-1ra) increased significantly two days before the clinical diagnosis of sepsis.10 In contrast to CRP, IL-6 is a very early marker, but levels can become normal even if infection continues. This leads to an increasing false negative findings when sample is performed later in the course. The simultaneous determination of CRP can obviate this problem, because the rise in plasma CRP levels occurs 12 to 48 hours after the onset of infection, at a time when IL-6 levels probably would have fallen. When these two markers are combined, the sensitivity becomes 100%.11,12,13 A semi-quantitative IL-6 quickest is available that provides accurate results within 15 minutes.
In addition to the proinflammatory endothelial and phagocytic activations, anti inflammatory cytokines also increase during sepsis. A major focus of current research is IL-10 which strongly inhibits the inflammatory cytokines TNF-a, IL-1, IL-6,IL-12 and IL-18.11,12,13

Granulocyte Colony Stimulating Factor (G-CSF) :
G-CSF is a haematopoietic growth factor that plays a pivotal role in promoting the growth and differentiation of granulocyte precursors and increasing the functional activities of their mature progeny. G-CSF production occurs in several type of cells such as monocytes, macrophages, epithelial cells and fibroblasts. Polysaccharide is a major stimulus for G-CSF production in neonatal sepsis. Several studies indicate G-CSF has 95% sensitivity for prediction of sepsis and a specificity of 73% for levels of more than 200 pg/ml.3,13,14

C-Reactive Protein (CRP) :
Physiology and Measurement :
C-Reactive Protein (CRP) is so called because it can be precipitated by the somatic C Polysaccharide streptococcus pneumoniae.15 It is produced by hepatocytes. Its concentration is less than 1 mg/dL in healthy individuals. It can rise 1000 fold within 24 hours under conditions characterized by inflammation with tissue destruction, especially bacterial infection.1,2,11,12 Although a specific primary role of CRP is uncertain, it has been shown to have multiple effects on immune systems and can activate complement. It is not influenced by serum proteins and RBC chracteristics like ESR. Any tissue destroying event, however, such as trauma, burn injury, ischemia or infarction can elicit production of this acute phase reactant. CRP is degraded rapidly, having half life less than 24 hours. Therefore, serial measurements can provide additional information on the adequacy of treatment.
Extremely high concentrations of CRP can be present in serum, yielding false negative test results if only undiluted serum specimen is used. Latex agglutination, immuno diffusion, enzyme immunoassay and nephelometry like various methods have been used for its estimation. Accurate measurement of CRP can be made by laser nephelometry or single radio-immunodiffusion assay. A semiquantitative bedside latex agglutination technique gives results within 15 minutes by rising capillary blood sample.4

Clinical Usefulness :
A number of acute phase proteins serve as useful indicators of infection in the neonates. The best studied among them is the CRP. In the first 4 days of life, apparently healthy neonates can have mildly elevated CRP (upto 1.5 mg/dL). Non infectious perinatal conditions like meconium aspiration, birth asphyxia, cephalhematoma and chest tube placement can cause elevated CRP, limiting usefulness of the test for prediction of neonatal sepsis. Its positive predictive value for septicemia is less than 50%. In these circumstances, it is not justified to treat the neonate with antibiotics only on the ground of positive CRP.4,11 Its negative predictive value for infection exceeds 98%. Very ill neonates may fail to mount a CRP response occasionally. In conclusion, negative CRP response almost excludes neonatal sepsis, but positive response needs correlation with other parameters as well as clinical picture and exclusion of other perinatal conditions.
CRP starts to rise within 12 to 24 hours of onset of sepsis, earlier than the other acute phase reactants. In a suspected case of sepsis if CRP is negative, it should be repeated alter 12 hours. A semi-quantitative bedside latex agglutination technique gives results within 15 minutes by using capillary blood sample. A qualitative assay of CRP does not offer significant advantages on the other indices. On the other hand, quantitative CRP values, particularly when repeated, are more useful.15,16,17,18,19,20
Because of rapid degradation of CRP, monitoring serial values can provide more information. Serial decline in CRP levels with therapy is suggestive of adequate response to antibiotics and recover)'. Usually, it returns to normal within 2 to 7 days of successful treatment. Persistent elevation of CRP may indicate persistent bacterial infection or development of some complication. Persistent elevation of CRP has been found with persistent bacterial meningitis and abscess formation in necrotizing enterocolitis. Serial CRP levels are useful in monitoring the response to treatment for osteomyelitis or septic arthritis.22,23
In one of the studies, progressively lower values from fourth to sixth day of therapy distinguished children with uneventful cases of treatment for acute osteomyelitis from these with complications.22 ESR remained elevated in all during the first week. CRP estimation in CSF is of no value for diagnosis of bacterial meningitis.23
CRP as a marker of cytokine release and active inflammation can be useful in diagnosis and management of many noninfectious inflammatory disorders such as Crohn's disease, rheumatoid disorders, autoimmune vasculitis diseases, Kawasaki disease, pancreatitis etc. Neoplasms of liver, lymphoma, allograft rejection and graft-versus-host disease are other conditions associated with high CRP values.

Procalcitonin (PCT) :
Procalcitonin is a propeptide of calcitonin, released into the blood 3 to 6 hours after endotoxin injection and increases upto 24 hours.3,24 The increase of procalcitonin does not correlate with calcitonin levels and occurs even in subjects who had thyroidectomy. It is physiologically elevated during first 3 days of life. But it has been found to be a reliable marker of late onset sepsis in newborn babies with a sensitivity and specificity of nearly 100%.25,2,29 The comparative studies have shown that PCT is a more reliable marker of sepsis compared to CRP.27 Quantitative measurement of PCT is performed, using an immunoluminometric assay (ILMA) with two monoclonal antibodies. After the initial surge of PCT during first 3 days of life, the mean normal serum PCT level is around 2 ng/ml.28 The possibility of bacterial infection and recommendation of antibiotics with reference to serum procalcitonin level can be predicted as shown in Table-III.
Table-III
Possibility of bacterial infection and recommendation of antibiotics with reference to procalcitonin level



Sensitivity and specificity of various markers of infection is shown in Table-IV with their comparison. It is worth to note their utility in clinical practice.

Table-IV
Comparison of various inflammation markers in clinical use


REFERENCES :
1. Kumar V, Abbas AK, Fausto N : Acute and chronic inflammation. Robbins and Cotran Pathologic basis of disease. 7th edn. New Delhi : Elsevier, a division of Reed Elsevies India Pvt. Ltd. 2007; 47-85. Long SS Laboratory Manifestations of Infectious Diseases In : Long SS, Pickering LK, Prober CG (Eds) Principles and Practice of Pediatric Infectious Diseases, New York : Churchill Livingstone 1997; 1530-1570.
2. Katewa S, Bansal D, Sachdeva A, Yadav SP : Markers for sepsis diagnosis : do they help? In : Selected topics in Pediatrics. Pedicon, 2008, 17th – 20th January, 2008, Orissa. 2008 ; 45-60.
3. Singh M : Care of the Newborn (6th edn), New Delhi : Sagar Publication 2004; 212.
4. Isaacs D, Moxon ER. Neonatal Infections IstIndian Edn 1994; 39.
5. The sepsis screen in the newborn deserves a decent burial – A Debate Dr. Gaetano Chirico says, “Yes”, Dr. Siddharth Ramji says, “No”. Presented at CME on Neonatal Sepsis at PGMER, Chandigarh.
6. Malhotra S, Sachdeva A, Yadav SP, Kler N, Saluja S, Soni A : Role of
cytokines in diagnosis of neonatal sepsis In : Selected topics in pediatrics. Pedicon, 2008. 17th-20th January, 2008, Orissa, 2008; 61-66.
7. Buck C, Bundschu J, Gallati H, et al : Interleukin-6 : a sensitive parameter for the early diagnosis of neonatal bacterial infection. Pediatrics 1994; 93 : 54-8.
8. Messer J, Eyer D. Donato L, et al : Evaluation of interleukin-6 and soluble receptors of tumour necrosis factor for early diagnosis of neonatal infection. J Pediatr 1996; 129 : 574-80.
9. Kuster H, Weiss M, Willeitner AE, Detlefsen S, Jeremias I, Zbojan J, et al : Interleukin-1 receptor antagonist and interleukin-6 for early diagnosis of neonatal sepsis 2 days before clinical manifestation. Lancet 1998 352 : 1271- 1277.
10. Puopolo KM. Bacterial and Fungal Infections In : Manual of Neonatal Care. Cloherty JP, Eichenwald EC, Stark AR (Eds) New Delhi, Wolters Kluwer (India) Pvt. Ltd. 2008; 274-300.
11. Gabay C, Kushner I : Acute phase proteins and other systemic responses to inflammation. N Engl J Med 1999 ; 340-445.
12. Mehr S, Doyle LW : Cytokines as markers of bacterial sepsis in newborn infants : a review. Pediatr Infect Dis J 2000; 19 : 879-887.
13. Berner R, Niemeyer CM, Leititis JU, et al : Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor- alpha, interleukin (IL)-1-beta and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis. Pediatr Res 1998; 44 : 469-77.
14. Prajapati BS, Prajapati RB, Patel PS : C-Reactive Protein : Current Perspectives. Bulletin ID Chapter of IAP.
15. Ng PC, Cheng SH, Chui KM, et al : Diagnosis of late onset neonatal sepsis with cytokines, adhesion molecules, and C-reactive protein in preterm very low birth weight infants. Arch Dis. Child Fetal Neonatal Ed 1997; 77 : F221- 7.
16. Berger C, Uehlinger J, Ghelfi D, et al : Comparison of C-reactive protein and white blood cell count with differential in neonates at risk of septicaemia. Eur J Pediatr 1995; 154 : 138-44.
17. Da Silva O, Ohlsson A, Kenyon C. Accuracy of leukocyte indices and C- reactive protein for diagnosis of neonatal sepsis : a critical review. Pediatr Infect Dis J 1995; 14 ; 362-6.
18. Laborada G, Rego M, Jain A, Guliano M, Stavola J. : Diagnostic value of cytokines and C-reactive proteini n the first 24 hours of neonatal sepsis. Amer J Perinatol 2003; 20 : 491-502.
19. Young B, Gleeson M, Cripps AW : C-reactive protein : a critical review Pathology 23 : 118-24, 1991.
20. Hanson LA, Jodal U, Sabel KG et al : The diagnostic value of C-reactive protein Pediatr Infect D J 2 : 87-90, 1983.
21. Ronie I, Faingezicht I, Argnedas A et al : Serial Serum C-reactive protein to monitor recovery from acute hematogenous osteomyelitis in children. Pediatr Infect. Dis J 14 : 40-4, 1995.
22. Gerdes LU, Jorgensen PE, Nexo E, Wang P : C-Reactive Protein and bacterial meningitis a meta-analysis. Scand J Clin Invest 1998; 58 : 383-393.
23. Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, et al. : Procalcitonin increase after endotoxin injection in normal subjects. J Clin Endocrinol Metab 1994; 79 : 1605-1608.
24. Balol C, Sungurtekin H, Gurses E, Sungurtekin U, Kaptanoglu B. Usefulness of procalcitonin for diagnosis of sepsis in the intensive care unit. Crit Care 2003; 7 : 85-90.
25. Ugarte H, Silva E, Mercan D, De Mendonca A, Vincent JL. Procalcitonin used as a marker of infection in the intensive care unit. Crit. Care Med 1999 ; 27 : 498-504.
26. Luzzani A, Polati E, Dorizzi R, Rungatscher A, Patan R, Merlini A : Comparison of procalcitonin and C-reactive protein as markers of sepsis. Crit Care Med 2003 ; 31 : 1737-1741.
27. Chiesa C, Panero A, Rossi N, et al : Reliability of procalcitonin concentrations for the diagnosis of sepsis in critically ill neonates. Clin Infect Dis. 1998; 26 : 664-72.
28. Harbarth S, Holeckova K, Froidevaux C, Pitter D, Ricou B. Grau GE, et al : Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis. Am J Respir Crit Care Med 2001; 164 : 396-402.