Introduction

Neonatal sepsis is defined as an aggressive bacterial infection occurring in the first 4 weeks of life. The incidence of neonatal sepsis varies from 11-24.5 /1000 live births in India [1]. The clinical pointer of sepsis in new-born infants is usually non-specific.

Because of the high morbidity and mortality which is related to neonatal sepsis [2,3,4], antibiotic therapy is started soon after the commencement of the symptoms before the diagnosis is long-established by blood culture.

The use of effective antimicrobial therapy has evidently reduced neonatal mortality. However, there is a need for a rapid test that can identify infected neonates at the time of initial valuation thus sparing the uninfected ones from redundant antibiotic therapy.

The current practice of starting empirical antibiotic therapy in all neonates showing infection-like symptoms results in their exposure to adverse drug effects, nosocomial complications, and in the emergence of resistant strains. [5].

Sepsis results from the complex interaction between the invading microorganism and the host immune, inflammatory, and coagulation response. [6,7] Inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-15, IL-18, MIF) and growth factors (IL-3, CSFs), and their secondary mediators, including nitric oxide, thromboxanes, leukotrienes, platelet-activating factor, prostaglandins, and complement, cause activation of the coagulation cascade, the complement cascade, and the production of prostaglandins, leukotrienes, proteases, and oxidants. [8]

Laboratory sepsis markers represent a helpful tool in the evaluation of a child with clinical signs and complement the evaluation of a neonate with a potential infection. During the last decades' efforts were done to improve laboratory sepsis diagnosis and a variety of the above-mentioned markers and more were studied with different success.

Despite the promising results for some of them, the current evidence suggests that none of them can consistently diagnose 100% of infected cases.

C - reactive protein (CRP), Blood culture (BC), Hematological Scoring System (HSS) are three well-identified parameters indicated for analysis of Neonatal Sepsis or infection.

ratio are related to early onset of sepsis in infants. High or low white blood cell counts, high absolute neutrophil counts, high immature /total ratio and low platelet counts are related to the late onset of sepsis.

Though all of these mentioned parameters are linked with sepsis or infection, all of these values separately have low sensitivities. Monroe devised a criterion that used three parameters of total PMN count, immature PMN count and I: T ratio, whereas in this hematologic scoring system.

Here, the current study undertake the study to evaluate the performance of the hematological scoring system (HSS) of Rodwell et al. (1988) in 110 neonates for the early detection of sepsis in high-risk infants, which should improve the diagnostic accuracy of the complete blood cell count as a screening test [19,20].

The present study was conducted to review the role of HSS and CRP as a diagnostic parameter in neonatal infections. The purpose was to obtain a range of tests that are fast, efficient, feasible and practically possible for laboratories with small setups in the developing as well as developed countries for diagnosis of neonatal sepsis.

Material and Methods

Type of study: The current study was a prospective hospital-based cross-sectional study carried out at the Department of Pathology during months of March and April 2019.

Sample size: A total of 40 neonates suspected of having sepsis were enrolled in the current study

Inclusion criteria:

• Neonates with clinical features suggestive of sepsis.
• Neonates with recent maternal infections.

Exclusion criteria:

• Intraventricular hemorrhage
• Meconium aspiration
• Pneumothorax
• Those who underwent surgery
• Birth asphyxia
• Antibiotic therapy prior to admission

Study procedure

Relevant maternal and neonatal histories were

Band cells: ≥ 20%.

Platelet count ≤ 1, 50,000 cells/mm.³

Considered as a positive septic screen if any two or more of the above criteria are met.

Probable sepsis

If the septic screen was positive or clinically symptomatic but blood culture negative.

No sepsis

Sepsis screen, as well as blood culture, are negative and baby is asymptomatic

Statistical Analysis

The data obtained were tabulated on Microsoft excel spreadsheets and analyzed. The data were expressed in terms of Mean±SD and percentages.

Sensitivity, specificity, Positive Predictive Values (PPVs) and Negative Predictive Values (NPVs) were calculated for each parameter.

Table 1: Haematologic scoring system (HSS).

Hematological scoring system
Criteria	Abnormality	Score
Total leukocyte count (cells/cumm)	<5000	1
	>20,000
ANC (cells/cumm)	<1800	1
Immature neutrophil count (cells/cumm)	<1200	1
I:T	≥0.2	1
I:M	≥0.3	1
Platelet count (cells/cumm)	<150,000	1
Degenerative changes in neutrophils	Toxic granules cytoplasmic vacuoles	1
I: T – Immature to total neutrophils ratio; I: M – Immature to mature neutrophils ratio
ANC – absolute neutrophil count

Results

In the current study, blood culture was positive in 10 cases (25%), out of which 6 (60%) were Gram-positive and 4 (40%) were Gram-negative, whereas 30 cases were found to be negative for blood culture. If the bacteriological profile of the culture is assumed to be the standard outcome, 10 cases are labeled as the definitive sepsis cases.

Klebsiella Pneumonia, Pseudomonas, Enterococcus, Staphylococcus aureus,and E. coli were the infections observed in the positive culture (Table 2).

The current study showed 10 neonates with positive culture, of which all of them had a HSS of ≥6 (Table 7). The sensitivity, specificity, PPV and NPV of HSS with cut-off score of 6 in predicting sepsis was 33.3%, 56.6%, 62.5%, 100%.

Similarly, the sensitivity, specificity, PPV and NPV of HSS with cut-off score of 5 in predicting sepsis was 33.3%, 40%, 40%, 100%. , the sensitivity, specificity, PPV and NPV of HSS with cut-off score of 4, 3 and 2 in predicting sepsis was 33.3%, 30%, 32.2%, 33.3%, 13.3%, 27%, 33.3%, 6.66%, 25% (Table 8).

Table 7: Diagnostic Accuracy of HSS.

HSS	Positive Culture	Negative Culture	Total
≥6	10	6	16
<6	-	17	17
≥ 5	10	15	25
<5	-	12	12
≥ 4	10	21	31
<4	-	9	9
≥3	10	27	37
<3	-	4	4
≥2	10	29	39
<2	-	2	2
Total	10	30	40

Table 8: Diagnostic accuracy of different haematological scores.

HSS	Sensitivity	Specificity	PPV	NPV
HSS > 6	33.3%	56.6	62.5	100
HSS > 5	33.3%	40	40	100
HSS > 4	33.3%	30%	32.2	100
HSS > 3	33.3%	13.3	27	100
HSS > 2	33.3%	6.66%	25	100

Out of the 10 positive cultured samples from the neonates, 3 had normal I: T Ratio and 7 had increased I: T Ratio. The Sensitivity, Specificity, PPV, and NPV of increased I: T Ratio in predicting sepsis was observed to be 70%, 100%, 100%, 90.0%.

The number of neonates that had increased PMN count was 10, with Sensitivity, Specificity, PPV, and NPV of increased PMN Count in predicting sepsis was observed to be 100%, 13.3%, 27.77, 100%.

In the current study, 7 neonates had increased I: T Ratio with Sensitivity, Specificity, PPV, and NPV of increased I: T Ratio in predicting sepsis was observed to be 70%, 100%, 100%, 90.0%.

The number of neonates with increased Total WBC Count was found to be 10 with Sensitivity,

the two parameters represented the significant relationship. 10 cases of the positive culture with the mean CRP value of 85.9±1.27 had an HSS of ≥5, whereas 4 cases of the negative group with the mean CRP value of 7.52±2.34 had HSS of 0-2, 11 cases of the negative group with the mean CRP of 25.21±3.21 had HSS of 3-4, lastly 15 cases of the negative group with the mean CRP of 56.24±3.81 had HSS of ≥5.

Table 11: Haematological score in comparison with CRP value.

HSS	C Reactive Protein (Mean Value)		Total cases
	Positive	Negative
0-2	0	7.52±2.34	4
3-4	0	25.21±3.21	11
≥5	85.9±1.27	56.24±3.81	15
Total	10	30	40

Figure 1 represented Leucocytosis with degenerative changes and band forms. Immature polymorphs include promyelocyte, myelocyte, metamyelocytes, and band forms.

Band cell is described as a PMN in which the nucleus is indented by more than one‑half, but in which, the isthmus between the lobes is wide enough to reveal two distinct margins with nuclear material in between.

Vacuolization, toxic granulations, and Dohle bodies comprise of degenerative changes.

Fig-1: Leucocytosis with degenerative changes and band forms.

Toxic granulations represent the cytoplasmic alterations in peripheral blood neutrophils in response to bacterial infection and have been found to be of greater use in differentiating localized from generalized infection or the development of complications (Figure 2).

complement system. An activated complement system induces granulocyte, phagocyte, and proinflammatory cytokine production. C-reactive protein is an acute-phase protein synthesized along with the activation of proinflammatory cytokines. The same thing will happen to granulocyte which is also a component of white blood cells [22]. The wide variability in diagnostic values of CRP may have been influenced by sample characteristics, study design, sample size, inclusion criteria, and differences in CRP cut-off points. C-reactive protein levels increase 24 to 48 hours after clinical manifestations appear. In the current study, the mean values of CRP were compared with the positive culture cases and negative culture cases with that of the control cases. The overall comparison was further linked to the reference range. Jave DL [23] stated that monitoring of CRP over time may be used to in determining the response of the treatment after the primary diagnosis. They were discharged home after 5 days of intravenous antibiotic therapy. Jin Cherdze and colleagues [24,25] concluded in their study that quantitative CRP is a rapid, sensitive diagnostic marker for the identification of sepsis in preterm infants. In the current study, it was observed that CRP is a good indicator of neonatal sepsis as the quantitative status of CRP helped in the identification of neonatal sepsis and also in deciding the line of management of the patient.

The common age distribution in the current study was between 6-10 days and ≤5 days in both the positive and negative culture cases. 6 (60%) of positive culture cases were under 6-10 days while 13 (43.3%) of cases were ≤ 5 days. Several studies reported age to be significantly associated with neonatal sepsis [26,27,28]. Based on Rodwell’s scoring system, neonates were classified as sepsis to be unlikely in 4 cases, possible in 11 cases and very likely in 25 cases. In the current study considering all four parameters i.e. sensitivity, specificity, positive predictive value and negative predictive value, I: T PMN ratio and degenerative changes were the most reliable tests for diagnosing sepsis. An abnormal I: M PMN ratio was highly sensitive in identifying sepsis. Degenerative changes in neutrophils were not found to be a very sensitive indicator of sepsis. Thrombocytopenia was consistently associated with poor prognosis. These findings were in comparison with other studies [29-32]. The higher the score, the greater was the likelihood of sepsis. A score ≤2 suggests that sepsis was unlikely.

Author’s contribution

All the authors, Dr. Chandrahas Ramesh Godbole, Dr. Sneha Ramdas Joshi, and Dr. Janice Jaison contributed equally in the conduct of the study and in the preparation of the manuscript.

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