|Year : 2012 | Volume
| Issue : 3 | Page : 97-100
Neonatal sepsis - Role of certain immunohematologic tests in rapid diagnosis
Santosh Kumar Mondal1, Dipanwita Roy Nag1, Ranjana Banyopadhyay2, Debdutta Chakraborty3, Swapan Kumar Sinha1
1 Department of Pathology, Medical College, Kolkata, West Bengal, India
2 Department of Pathology, Burdwan Medical College, Kolkata, West Bengal, India
3 Department of Community Medicine, Medical College, Kolkata, West Bengal, India
|Date of Web Publication||21-May-2014|
Dr. Santosh Kumar Mondal
"Teenkanya Complex", Flat 1B, Block B, 204 R N Guha Road, Dumdum, Kolkata - 700 028, West Bengal
Source of Support: None, Conflict of Interest: None
Background and Objective : Many neonates with sepsis die worldwide due to lack of early diagnosis and prompt treatment. This study was carried out to analyze various immunological and hematological parameters singly and in combination to formulate a guideline for the rapid diagnosis of neonatal sepsis.
Materials and Methods : In a prospective study, 62 patients having clinical suspicion of neonatal sepsis were evaluated with a battery of investigations. Neonates admitted for other causes and without clinical suspicion of sepsis were selected as controls (n = 40). The tests included blood culture, hemoglobin (Hb) level, total and differential blood count, absolute neutrophil count (ANC), immature to total neutrophil count (I/T ratio), micro-erythrocyte sedimentation rate (m-ESR), C-reactive protein (CRP), platelet count, serum immunoglobulin M (IgM), and plasma fibrinogen level. Patients were divided into proven cases (positive blood culture) and probable cases (blood culture negative).
Results : Positive blood culture was seen in 38 cases (61.3%). Raised m-ESR (>8 mm in 1 st h) was seen in 63.2% of proven and 66.7% of probable cases. An I/T ratio of ≥0.2 was seen in 63.2% and 58.3% of proven and probable cases. Morphological changes were detected in 68.4% of proved and 91.7% of probable cases. Positive CRP test (≥6 mg/L) was found in 84.2% of proven and 100% of probable cases. Raised serum IgM, leukopenia, and neutropenia were seen in minority of patients (11-37%). Raised fibrinogen (>400 mg/L) was seen in the patients as well as in controls.
Conclusions : Four useful tests were m-ESR, I/T ratio, morphological changes in neutrophils, and CRP; and were statistically significant (P < 0.05). Most sensitive test was CRP (84%) and m-ESR was most specific test (94%). Combination of three or more of these four tests were highly specific (95-100%).
Keywords: Laboratory tests, neonatal sepsis, rapid diagnosis
|How to cite this article:|
Mondal SK, Nag DR, Banyopadhyay R, Chakraborty D, Sinha SK. Neonatal sepsis - Role of certain immunohematologic tests in rapid diagnosis. J Acad Med Sci 2012;2:97-100
|How to cite this URL:|
Mondal SK, Nag DR, Banyopadhyay R, Chakraborty D, Sinha SK. Neonatal sepsis - Role of certain immunohematologic tests in rapid diagnosis. J Acad Med Sci [serial online] 2012 [cited 2020 Sep 25];2:97-100. Available from: http://www.e-jams.org/text.asp?2012/2/3/97/132949
| Introduction|| |
Neonatal sepsis is a clinical syndrome resulting from the pathophysiological effect of severe bacterial infection in the 1 st month of life. In contrast to bacteremia (bacteria in blood), septicemia usually consists of bacteremia plus a constellation of signs and symptoms caused by microorganisms or their toxic products in circulation. There may be progression of bacteremia to septicemia depending on clinical manifestations. However, septicemia may also occur without bacteria, such as culture negative sepsis associated with pyelonephritis or pneumonia due to endotoxemia.
Clinical diagnosis of neonatal sepsis is difficult because signs and symptoms are not always specific. There is no laboratory test with 100% sensitivity and specificity. , Blood culture has been considered as gold standard for confirmation of diagnosis but these results are available only after 48-72 h. Moreover, in many cases blood culture fails to detect the offending organism/bacteria. So, search is still continuing for a reliable test, especially in culture negative cases.
| Materials and Methods|| |
A total of 62 neonates (infant <1 month of age) with clinical suspicion of septicemia were selected in a prospective study over a period of 1 year, from June 2008 to May 2009 in our institute. Septicemia was suspected from clinical history, signs, symptoms, and predisposing factors in mothers and neonates. Maternal factors included illness during pregnancy like hypertension, chronic renal disease, diabetes mellitus, toxemia of pregnancy, antepartum hemorrhage, maternal fever during last 2 weeks of pregnancy, prolonged rupture of membranes (>24 h), meconium stained liquor amnii, and difficult labor with instrumentation. Neonatal factors included artificial resuscitation, intravenous fluid administration umbilical vein catheterization, failure to thrive, refusal to suck, abdominal distension, jaundice, recurrent attacks of apnea and cyanosis, hypothermia, sclerema, and convulsion.
Before collection of blood, puncture site and adjacent area were sterilized. Blood was collected by femoral vein puncture and transferred into culture broth and into three different vials for different hematological and immunological tests. Ethylenediaminetetraacetic acid (EDTA) mixed venous blood was used to estimate hemoglobin (Hb), total leukocyte count (TLC), and platelet count. Citrated blood was used to estimate plasma fibrinogen level. For estimation of serum immunoglobuin M (IgM) and CRP, clotted blood was used. To estimate micro-erythrocyte sedimentation rate (m-ESR), capillary blood was taken in a heparinized micro-hematocrit tube. For differential leukocyte count, absolute neutrophil count (ANC), morphological changes in neutrophils, and I/T ratio; blood smears were made on glass slides. Plasma fibrinogen was estimated by reagent kit which was based on modified Clasus method. Diagnostic kit was also used to determine serum IgM by an immunochemical assay. After obtaining test results, statistical analysis (P value, Chi-square test (χ2 ), and degree of freedom) was made using Statistical Package for Social Sciences (SPSS) software, version 17.
Among the 62 clinically suspected cases, blood culture was positive in 38 cases and regarded as 'proven' cases. In 24 cases, though the blood culture report was negative, there were strong clinical suspicion and later supported by other laboratory tests. This second group was regarded as 'probable' cases.
| Results|| |
In the present study, 38 out of 62 cases had positive blood culture reports. Gram negative organisms are commoner than gram positive organisms (68.4 vs 31.6%). Klebsiella pneumoniae was the commonest bacteria (52%), followed by Staphylococcus aureus (26%). In one case there was mixed infection of K. pneumoniae and S. aureus. Twelve out of 26 neonates suffering from gram negative bacteria, and four out of 12 neonates suffering from gram positive bacteria died in spite of treatment [Table 1]. Among the proven cases, 32 out of 38 cases (84%) had low birth weight (LBW; weight <2.5 kg), whereas 37% (14/38) of these neonates were preterm (<37 weeks of gestational age).
Among all the tests performed, four tests (CRP, m-ESR, I/T ratio, and morphological changes in the neutrophils) showed good results to diagnose the cases and were statistically significant [Table 2]. m-ESR test had highest specificity (94%) and CRP test had highest sensitivity (84%). Highest positive predictive accuracy was seen in m-ESR (92%) [Table 3]. But when combination of these tests was taken together, specificity and positive predictive accuracy increased. When two tests became positive, specificity and positive predictive accuracy were 84 and 85%, respectively. When three tests had positive results specificity and positive predictive accuracy were 88 and 95%, respectively. But if the all four tests became positive, then both specificity and positive predictive accuracy were 100%. Best combination of two tests in this study found to be CRP + I/T ratio (sensitivity 52% and specificity 95%). Second best combination was m-ESR + CRP, followed by m-ESR + I/T ratio. But, when combination of these tests were considered, sensitivity decreased (84% in two positive tests, 42% in three positive tests, and 21% in four positive tests).
|Table 2: Important four tests and their results in control, probable, and proven cases|
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|Table 3: Sensitivity, specificity, positive predictive accuracy of the above tests, and their different combinations|
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Out of 38 proven cases, 27 cases (69.1%) had low Hb (<17 g/dL) and 22 of them had unconjugated hyperbilirubinemia which was neither due to Rh hemolytic disease or ABO blood group incompatibility [Table 4]. Among the probable cases, 14 out of 24 cases (58.3%) had low Hb, and 10 cases had unconjugated hyperbilirubinemia. Leukopenia was seen in 4/38 proven cases on 'Day 4' investigation and all of them died. Only two patients developed leukocytosis on 'Day 4' investigation and they survived. In the probable cases of sepsis, the findings were similar. In both proven and probable cases of sepsis, the percentage of neonates showing thrombocytopenia was higher than control cases. High fibrinogen level was found in 4/24 probable cases (16.6%) and in 9/38 proved cases (23.7%) and one in control cases. Raised serum IgM level was seen in the majority of proved and probable cases as well as in controls.
|Table 4: Results of other tests in control, probable, and proven sepsis cases|
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| Discussion|| |
The World Health Organization estimates that perinatal cases are the commonest cause of childhood mortality below the age of 5 years in developing countries.  Neonatal infections are by far the most common cause of such perinatal mortality. ,
In the present study, majority of neonates with proven sepsis (32/38 or 84.2%) had LBW and a fair number of them were preterm (14/38 or 36.8%). Ng et al., used CRP, cytokines, and adhesion molecule to identify late onset neonatal sepsis (LONS) in preterm very LBW infants. They found interleukein-6 (IL-6) had the highest sensitivity (89%) and negative predictive value (NPV; 91%) for detecting infection on 'Day 0'. However, between 24 and 48 h of onset, CRP was the best single marker. In another study, it was found that IL-6 combined with procalcitonn values was a fair way to evaluate early onset neonatal sepsis (EONS). But I/T ratio was as efficient as IL-6.  Walliullah et al.,  reported sensitivity and specificity of m-ESR (63.3 and 60%, respectively) and I/T ratio (70 and 56%, respectively) in neonatal sepsis. Combination of m-ESR and I/T ratio showed high sensitivity (80%) and specificity (70%). But in the present study, combination of m-ESR and I/T ratio showed decreased sensitivity (31%) and high specificity (95%). Several other authors used CRP and hematological parameters in neonatal sepsis. ,, Most of them found that CRP and specially a combination of these tests were very helpful to diagnose neonatal sepsis. During the first 3 days of life; CRP, leukopenia, and neutropenia were comparatively good tests, while after 3 days of life CRP was the best single test in early detection neonatal sepsis.  In our study, CRP was the most sensitive test (84%) with a specificity of 65%. But when CRP was combined with I/T ratio, specificity increased to 95%. Combination of CRP with other tests also gave good results. CRP might be used for differentiation between positive and contaminated blood culture in children and has shown to be a better predictor than white blood cell (WBC) or ANC for this purpose.  Morphological changes (toxic granules, vacuoles, and Dohle bodies in the cytoplasm of neutrophils) though had a high sensitivity (68%) and specificity (80%) in our study, but others found lower sensitivity (44%) and higher specificity (94%).  Some authors formulated a hematological scoring system (HSS) to diagnose EONS. , A hematological score of ≥ 3 had a sensitivity of 86% and NPV of 96%. 
Use of hematological tests like Hb estimation, TLC, differential leukocyte count (DLC), and platelet count; when combined with the four important tests mentioned earlier, found to be very useful to diagnose neonatal sepsis. ,, In this study, 27/38 (69.1%) had low Hb and many of them (18/22) had unconjugated hyperbilirubinemia which was not due to ABO or Rh incompatibility. In these cases low Hb level could be due to increased hemolysis of RBC caused by bacterial infection in blood.
The first Immunoglobulin (Ig) produced by neonates is IgM and increased level of this antibody is found in acute bacterial, viral, and parasitic infections. In this study, serum IgM was raised in both proven sepsis cases and controls. So, the test was not very helpful in diagnosing neonatal sepsis. Similar observation was found by Adhikari et al., in 1986.  But others found that IgM concentration in the sepsis group (median 34 mg/dL) was significantly higher than that in the 'no sepsis group' (median 10 mg/dL; P < 0.0001).  Death rate among neonates with IgM levels <20 mg/dL was five times higher than those with elevated IgM levels.  In the present study, fibrinogen levels were not a helpful test to identify neonatal infection. Speer et al., made similar observations.  Recently, molecular analysis by polymerase chain reaction (PCR) for bacterial DNA component encoding 16s RNA has been found to be very useful and superior to blood culture for early diagnosis of sepsis in neonates. PCR has a sensitivity of 100% and specificity of 95.6%. 
| References|| |
|1.||Plazek MM, Whitelaw A. Early and late neonatal septicemia. Arch Dis Child 1983;58:728- 31. |
|2.||Gluck L, Wood HF, Fousek MD. Septicemia of the newborn. Pediatr Clin North Am 1966;13:1131-48. |
|3.||WHO: World Health Report, Geneva: World Health Organization; 2005. |
|4.||Largo MG, Stekelenburg J. The millennium project of the United Nations, focusing on adequate postpartum care to reduce maternal and neonatal mortality world-wide. Ned Tijdschr Geneeskd 2006;150:1143-7. |
|5.||Mufti P, Setna F, Nazir K. Early neonatal mortality: Effects of interventions on survival of low birth babies weighing 1000-2000g. J Pak Med Assoc 2006;56:174-6. |
|6.||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;55:219-33. |
|7.||Walliullah SM, Islam SM, Siddika M, Hossain MA, Chowdhury AK. Role of micro-ESR and I/T ratio in the early diagnosis of neonatal sepsis. Mymensing Med J 2009;18:56-61. |
|8.||Ahmed Z, Ghafoor T, Waquar T, Ali S, Aziz S, Mahmud S. Diagnostic value of C-reactive protein and haematological parameters in neonatal sepsis. J Coll Physicians Surg Pak 2005;15:152-6. |
|9.||Berger C, Uehlinger J, Ghelfi D, Blau N, Fanconi S. Comparison of C-reactive protein and white blood cell count with differential in neonates at risk for septicaemia. Eur J Pediatr 1995;154:138-44. |
|10.||Manucha V, Rusia U, Sikka M, Faridi MM, Madan N. Utility of haematological parameters and C-reactive protein in the detection of neonatal sepsis. J Pediatr Child Health 2002;38:459-64. |
|11.||Shaoul R, Lahad A, Tamir A, Lanir A, Srugo I. C Reactive Protein (CRP) as a predictor for the bacteremia in children. Med Sci Monit 2008;14:CR255-61. |
|12.||Ghosh S, Mittal M, Jaganathan G. Early diagnosis of neonatal sepsis using a hematological scoring system. Indian J Med Sci 2001;55:495-500. |
|13.||Varsha, Rusia U, Sikka M, Faridi MM, Madon N. Validity of hematologic parameters in identification of early and late onset neonatal infection. Indian J Pathol Microbiol 2003;46:565-8. |
|14.||Selimovic A, Skokic F, Selimovic Z, Bazardzanovic M. The predictive values of total white blood count and differential count in the diagnosis of early-onset neonatal sepsis. Med Arch 2008;62:205-10. |
|15.||Adhikari M, Coovadia HM, Coovadia YM. Predictive value of C-reactive protein in neonatal septicemia. Ann Trop Pediatr 1986;6:37-40. |
|16.||Khassaweneh M, Hayajneh WA, Kofahi H, Khader Y, Amarian Z, Daoud A. Diadnostic markers for neonatal sepsis: Comparing C-reactive protein, interleukein-6 and immunoglobulin M. Scand J Immunol 2007;65:171-5. |
|17.||Krishna BV, Nadquir SD, Tallur SS. Immunoglobulin-M estimation and C-reactive protein detection in neonatal septicemia. Indian J Pathol Microbiol 2000;43:35-40. |
|18.||Speer CP, Gahr M, Schroter W. Early diagnosis of neonatal infection. Montsschr Kinderheilkd 1985;113:665-8. |
|19.||Yadav AK, Wilson CG, Prasad PL, Menon PK. Polymerase chain reaction in rapid diagnosis of neonatal sepsis. Indian Pediatr 2005;42:681-5. |
[Table 1], [Table 2], [Table 3], [Table 4]