THE VALIDITY OF HEMATOLOGIC MARKERS FOR DIAGNOSIS OF NEONATAL SEPSIS

Objective: To determine the validity of haematologic markers for sepsis screen (absolute neutrophil count (ANC), immature/total leukocytes ratio (I:T), platelets count (PC), C-reactive protein (CRP) and serum ferritin), both individually and in combination for early diagnosis of neonatal sepsis. Methodology: This cross – sectional analytical study was conducted in Neonatal Section of Paediatric Medicine Unit II, Mayo Hospital Lahore for one year. One hundred neonates presenting with clinical sepsis were included through non-probability, purposive sampling after written informed consent. Blood sample was collected for full hematologic screening as mentioned above along with blood cultures. Data was entered and analyzed using SPSS Version 17. Results: There were 68 male and 32 females including 31 preterm and 69 term neonates. 45 neonates were < 1 day age, 40 neonates 1 – 10 days age, 15 neonates Ayub A. Department of Paeds Medicine KEMU / Mayo Hospital, Lahore Chishti A.L. Chairman Department of Paeds Medicine KEMU / Mayo Hospital, Lahore Hassen K.A. Assistant Professor Department of Paeds Medicine KEMU / Mayo Hospital, Lahore 11 – 30 days age. Mean weight of study cases was 2.35 ± 0.69 Kg. Sensitivity, specificity and diagnostic accuracy of ANC were 77.3%, 100% and 90%, for I/T ratio were 81.8%, 81.4% and 81%, for CRP were 75%, 83.9% and 80%, for platelet count were 84.1%, 71.4% and 77%, for serum ferritin were 88.6%, 69.6% and 78% respectively. Sensitivity, specificity and diagnostic accuracy of combination of SF + I:T was 81.8%, 82.1% and 82%, for combination of SF + ANC + I:T were 93.2%, 71.4% and 81% for combination of SF + CRP + I:T were 93.2%, 67.9% and 79%, for combination of SF + I:T + PC were 90.9%, 58.9% and 73%, for combination of SF + CRP + ANC were 95.5%, 69.6% and 81%. Sensitivity, specificity and diagnostic accuracy of combination of all markers were 90.9%, 76.8% and 83% respectively. Conclusion: Results of our study showed that we can safely rely on hematologic markers for confirmation of neonatal sepsis both singly and in combination. We suggest that these tests may help to diagnose neonatal sepsis earlier.


Introduction
Neonatal Sepsis is a clinical syndrome characterized by signs and symptoms of infection with or without accompanying bacteraemia in the first month of life. 1 The incidence of neonatal sepsis in India has been Original Article estimated as 30 per 1000 livebirths. 2 The reported incidence of neonatal sepsis varies from 7.1 -38 per 1000 live births in Asia and from 3.5 -8.9 per 1000 live births in South America and Caribbean. 3,4The estimated mortality rate is as high as 30 % and in some studies up to 69%. 5 WHO estimates show that one million deaths occur every year due to the neonatal sepsis (10% of < 5 year mortality).Forty two percent of these deaths occur during 1 st week of life. 6,7The most important risk factors are prolonged rupture of membrane (PROM), maternal pyrexia, prematurity, low birth weight and prolonged hospitalization in the neonatal intensive care units. 3,8,9arly warning signs and symptoms are often nonspecific and can easily be confused with non-infective causes.The common clinical features of neonatal sepsis are lethargy, poor feeding, temperature instability and skin mottling.However, some of the neonates may manifest with life threatening involvement of cardiac and respiratory systems. 10,11ositive blood culture is considered gold standard for diagnosis of micro-biological infection.However, blood cultures are positive only in 5 -10% of suspected sepsis cases, even at highly resourced facilities because of earlier exposure to antibiotics before samples are withdrawn.Moreover, biological culture results are usually unavailable before 48 -72 hours.Hence a reliable infection marker or a set of markers are needed to promptly and accurately identify the infected cases. 11,12he simple hematologic screen (absolute neutronphil count, immature/total leukocyte ratio, platelet count, C-reactive protein and serum ferritin) are useful markers for the early diagnosis of neonatal sepsis.Local data suggests that CRP was positive in 80.5% of probable sepsis with a specificity of 95%.Another Egyptian study showed CRP as best laboratory test with an overall sensitivity and specificity of 86% and 97% respectively. 10,13,14otal and differential leukocyte counts did not show promising results in a study but thrombocytopenia was present in 50% cases of culture positive sepsis.Another study showed the sensitivity of platelet count in proven sepsis group was 64.3%.However, thrombocytopenia is a non specific indicator of infection.][15] Serum ferritin is considered as positive acute pha-se reactant.One study concluded that ferritin was raised in children with septic shock. 16While in another study it was shown that serum ferritin levels were higher in two groups with sepsis, but compared with the healthy group, the difference was statistically insignificant in newborns. 17

Operational Definitions
Clinical Sepsis: According to IMNCI, sepsis was defined as presence of any sign listed implying high suspicion of serious bacterial infection: (convulsions, respiratory rate > 60 breaths/min, severe chest indrawings, temperature > 37.7ºC or < 35.5ºC, lethargic or unconsciousness, not able to feed/sucking, bulging fontanel, pus draining from the ear, grunting /nasal flaring, redness around umbilicus).Proven sepsis was defined as patients with positive blood cultures were categorized as having proven sepsis.Patients with negative blood cultures but abnormal hematologic results suggestive of sepsis were categorized as probable sepsis.

Material and Methods
This crosssectional analytical study was carried out in Neonatal Section of Paediatric Medicine Unit II, Mayo Hospital, Lahore over one year (Jan -Dec 2013).One hundred neonates presenting with clinical sepsis were included with non-probability, purposive sampling.The sample size was calculated using WHO

Discussion
Early diagnosis of neonatal septicemia is difficult. 10he positive blood culture report which is a gold standard for the diagnosis of neonatal sepsis requires 48-72 hours of waiting time.Further, the yield of the blood culture is also low and in some hospitals, the facileties may not be available.Hence, for early identification, several screening tests and their usefulness, either individually or in combinations, have been reported.
Recently, few researchers suggested the need for the reassessment of the CBC.They did not use CRP as a screening tool and reported a higher sensitivity of the physical examination. 11,12imple blood screening tests such as absolute neutrophil count (ANC), immature to total leukocyte Ratio (I: T), platelet count (PC), C -reactive protein (CRP) and serum ferritin (SF) for early detection of neonatal septicaemia were studied.The ratio of early neonatal sepsis was higher than late onset sepsis.One study also reported that frequency of early neonatal sepsis (54.53%) was higher than late neonatal sepsis (45.57%), without statistical significance. 18.Regarding sex distribution, our results compare with another study which reported the proportion cases of neonatal sepsis was higher in male (60%) than female neonates. 19But another study reported that risk of developing neonatal sepsis was nearly equal in both genders (57% male and 43% female).However no statistically significant difference was found. 18Gender predisposition to sepsis remained controversial, while a study done in Winthrop University hospital showed no gender difference in the frequency of bacterial sepsis; while several other studies had shown that the frequency and even severity of bacterial sepsis was higher in males. 20One case series reported the frequency of preterm neonates developing neonatal sepsis being 88.3%. 19,21others of 83% neonates complained about baby's poor feeding.This may contribute towards developing neonatal sepsis as mother's milk is important source of immune factors in neonates.Poor feeding or refusal to feed remains an important symptom of neonatal sepsis.
For ANC, sensitivity, specificity, PPV and NPV were 77.3%, 100%, 100% and 84.8% respectively.The overall diagnostic accuracy of ANC was calculated as 90%.But one study reported sensitivity for ANC was little higher than that of our study 84%. 22or I/T ratio, sensitivity, specificity, PPV and NPV were 81.8%, 81.4%, 76.6% and 84.9% respectively.The overall diagnostic accuracy of I/T ratio was estimated as 81%.These results were bit different from other studies.One study reported the sensitivity and specificity of I/T ratio was 63% and 85% respectively. 23But another study reported the sensitivity, specificity, PPV and NPV for I/T ratio were 33%, 100%, 100% and 66% respectively (22 Thus conflicting results are observed between different studies and require more studies to confirm the validity of these markers. For CRP as sepsis indicator, the estimated sensitivity, specificity, PPV and NPV were 75%, 83.9%, 78.6% and 81% respectively.The overall diagnostic accuracy of CRP was calculated as 80%.One study reported the sensitivity and specificity of CRP was 84% and 65% respectively. 23r Platelet Count (PC), sensitivity, specificity, PPV and NPV were 84.1%, 71.4%, 69.8% and 85.1% respectively.The overall diagnostic accuracy of PC was calculated as 77%.But one study reported the sensitivity and specificity of PC 88% and 51.4% with an accuracy of 56%. 24Another study reported the sensitivity, specificity, PPV and NPV for PC were 48%, 96%, 91% and 71% respectively. 22 also performed estimation of serum ferritin (SF) levels and found the sensitivity, specificity, PPV and NPV to be 88.6%, 69.6%, 69.6% and 88.6% respectively.The overall diagnostic accuracy was calculated as 78%.One study reported the sensitivity and specificity of SF as 100% and 58%, respectively. 16rious combinations of blood markers in sepsis had been studied earlier 15 to determine the diagnostic accuracy in neonatal sepsis and found valid.We found that sensitivity, specificity, PPV and NPV of combination of ANC+ CRP+ I:T ratio were 86.4%, 32.1%, 50% and 75% respectively.The overall diagnostic accuracy was 56%.One study reported that I:T ratio+ ANC+CRP was the best combination which gave 88% sensitivity, 84% specificity, 86% PPV and 85% NPV. 19We found that sensitivity, specificity, PPV and NPV of combination of ANC+CRP+I:T+PC+SF were 90.9%, 76.8%, 75.5% and 91.5% respectively.The overall diagnostic accuracy was 83%.
The combination of different tests showed higher sensitivity 95.5% for a combination of SF+CRP+ANC followed by 93.2% for (SF+IT+ANC) and 93.2% for SF+IT+CRP.Diagnostic accuracy on the other hand was found higher 83% with combination of all 5 tests (ANC+CRP+SF+PC+IT) and 82% with (SF+IT).This was followed by 81% both with (SF+CRP+ANC) and (SF+IT+ANC).

Conclusion
Results showed that hematologic markers for diagnosis of neonatal sepsis were quite reliable and valid.These blood tests can be used either singly or in combination.Both SF and I:T ratio were reliable indicators for early diagnosis of neonatal sepsis when used singly.However, SF+CRP+ANC, SF+I:T+ANC and SF+ CRP+I:T had also proven to be reliable combination for diagnosis of neonatal sepsis.Our results are promising and suggest their judicious use.However by adding results from further studies will help make uniform recommendations to develop standard protocols.

Table 1 :
software by taking incidence 38/1000 with 5% level of significance and 3.8% margin of error.Inclusion Criteria were neonates (age 1 st -28 days of life) of either sex, having any of the IMNCI signs of clinical sepsis.Neonates weighing < 1000 gm, gestational age < 28 weeks, H/O prior use of antibiotics and major systemic congenital anomalies were excluded.One hundred neonates of both genders with clinical signs of sepsis presenting in Neonatal Section at Department of Paediatrics Unit II, Mayo Hospital, Lahore, were enrolled for the study after obtaining informed written consent from the parents.Information was collected on structured questionnaires that included age, sex, anthropometric data and systemic details.A full hematologic screening including absolute neutrophil count, imam-Baseline Characteristics of Study Newborns (n = 100).
ture / total leukocyte ratio, platelet count, CRP and serum ferritin along with blood cultures were obtained before the start of anti-microbial therapy.Samples were collected for blood complete picture including ANC, I/T ratio, platelet count and serum ferritin levels.Sample was collected for estimation of CRP by latex agglutination technique (quantitative).Under strict aseptic measures, 1.5 ml venous blood was collected in blood culture bottles.The laboratory investigations were done at Paediatric Pathology Laboratory under supervision of qualified hematologist and bio-chemist.PC was done in the CBC using Sysmax method in heparinized / oxalate containing blood.ANC was calculated using formula of TLC x Neutrophil count / 100.I:T ratio was done by direct blood smears, stained with Giemsa and band neutrophild were counted.SFResultsA total 100 neonates with clinical signs of sepsis were studied.Their sex, age, weight at admission and gestational age distribution is shown in Table1.85% neonates presented with early onset while 15% presented with late onset neonatal sepsis.There were

Table 3 :
Validity of Absolute Neutrophil Count Taking Blood Culture as Gold Standard.

Table 4 :
Validity of Immature/ Total Leukocyte Ratio Taking Blood Culture as Gold Standard.

Table 5 :
Validity of C-Reactive Protein Taking Blood Culture as Gold Standard.

Table 6 :
Validity of Platelet Count Taking Blood Culture as Gold Standard.

Table 7 :
Validity of Serum Ferritin Taking Blood Culture as Gold Standard.

Table 8 :
Validity of SF+I:T Taking Blood Culture as Gold Standard.

Table 9 :
Validity of SF+ANC+IT Taking Blood Culture as Gold Standard.

Table 11 :
Validity of SF+IT+PC Taking Blood Culture as Gold Standard.

Table 12 :
Validity of SF+CRP+ANC Taking Blood Culture as Gold Standard.

Table 13 :
Validity of Combination of All Tests Taking Blood Culture as Gold Standard.