I. INTRODUCTION

The neonatal period is vulnerable to infections and its impact can have long term effects. In this period more than $50\mathrm{\%}$ of deaths are reported among under five children. 1NNPD 2002 -2003 report had showed incidence of neonatal sepsis as 30/1000 live births. 2Sustainable Developmental Goals (SDG) 3 aims to reduce neonatal mortality to less than 12 deaths for 1000 live births by 2030. W. H. Ohas given guidelines that can be utilized to achieve SDG 3.

After prematurity, sepsis is considered as the second leading cause of mortality. Incidence of clinical sepsis very high in India. ${}^4$ Presence of signs and symptoms of infection within one month of birth are considered as neonatal sepsis. ${}^5$

Neonatal sepsis can be divided into Early-onset sepsis (EONS), and Late-onset sepsis (LONS). Early-onset sepsis occurs within the first 72 hours of life. Late-onset sepsis generally occurs after 3 days of birth. Mortality is more with EONS than LONS.[6] On time diagnosis and management of neonatal sepsis is very essential to prevent mortality. Blood culture is the confirmatory method of neonatal sepsis. But among all the neonatal sepsis, only 25 to $40\mathrm{\%}$ have culture positivity. ${}^7$ In developing countries like India, with the resource-limited setting and the delay in obtaining culture positivity report (up to 48 hrs), confirmation of neonatal sepsis can be done based on the clinical presentation, and use of sepsis markers. Hence, this study was taken up to prove the diagnostic significance of sepsis markers in neonatal sepsis.

II. MATERIALS & METHODS

This is an observational study done from May 2020 to November 2021 at GEMS NICU, Ragolu, Srikakulam.

a) Inclusion Criteria

All babies days of life with sepsis risk factors and clinical features suggesting sepsis were included in this study.

b) Exclusion Criteria

Babies with birth asphyxia, with birth weight grams, with gestational age weeks and neonates who had taken antibiotics were excluded from the study.

In this observational study, neonates clinically suspected to have sepsis were enrolled. On meeting the inclusion criteria, prior informed, written parental consent was obtained before enrollment in the study. The study has been cleared by Hospital Ethics Committee and Hospital Research Committee. During the study, a pre-designed and pre-tested proforma was implemented.

On admission, as per the hospital protocol, detailed information was noted in the medical case sheet. When sepsis was suspected, clinical features were noted. Investigations like complete blood picture, CRP, and blood culture were done. Various biomarkers are used as diagnostic tools for neonatal sepsis. But the gold standard for the detection of sepsis is blood culture, but blood culture takes 2-3 days for a diagnosis.

This study evaluates the Sensitivity, specificity, positive predictive value, and negative predictive value of the sepsis markers i.e., Total Leukocyte Count, CRP, Platelet count. Data was analyzed with Microsoft Excel and SPSS (Statistical package for social sciences) version 25. Statistical analysis was done by using the Chi-square test and ANOVA. Results were expressed in terms of mean, percentages, and depicted as tables, graphs. P-value was considered statistically significant.

III. RESULTS

All the 100 neonates enrolled were survived without any mortality.

Of the 100 neonates, males were $63\mathrm{\%}$, and females were $37\mathrm{\%}$.

The average birth weight of preterm neonates was $2336.76\pm 148.23$ grams, and term neonates were

2661.03 ± 129.16 grams. The average birth weight of all (100) neonates was 2430.80 ± 205.238 grams.

The average gestational age of the preterm neonates was $34.29\pm 0.67$ weeks, term neonates were $37.88\pm 0.73$ weeks, and the average gestational age of total neonates was $35.65\pm 1.88$ weeks.

Of the total neonates, $57\mathrm{\%}$ were delivered by caesarean section and $43\mathrm{\%}$ were delivered by normal vaginal delivery (NVD).

In this study, regarding indication for caesarean section, foetal distress was the most often reason $(16\mathrm{\%})$ followed by meconium stained liquor $(12\mathrm{\%})$, CPD $(10\mathrm{\%})$,

Non-reactive NST (9%), non-progression of labour (8%), and compound presentation (2%) were the other causes for indication of caesarean section.

In this study regarding the distribution of risk factors for sepsis, $20\mathrm{\%}$ of the cases had single unclean or more than 3 Vaginal Examinations, $34\mathrm{\%}$ of cases had PROM more than 18 hrs, $21\mathrm{\%}$ of cases had meconium-stained liquor, $17\mathrm{\%}$ of cases had a febrile illness in mother, and $8\mathrm{\%}$ of the cases had foul Smelling Liquor.

Table 3:Manifestation of Sepsis

In this study, the majority (82%) of the neonates had a shock, followed by congenital pneumonia (9%).

In this study, early onset of neonatal sepsis (EONS) was observed among $56\mathrm{\%}$ of the neonates, and late-onset of neonatal sepsis (LONS) was observed among $44\mathrm{\%}$ of neonates.

The average duration of EONS was $18\pm 9.12$ hrs, and LONS was $168.1\pm 74.35$ hrs.

In this study, of all the neonates, $68\mathrm{\%}$ of the neonates had normal WBC levels and $32\mathrm{\%}$ of cases had abnormal WBC levels.

In this study, of the total neonates, $56\mathrm{\%}$ of the neonates had Lakhs platelets and $44\mathrm{\%}$ of neonates had $\le 1.5$ Lakhs platelets.

Among the total neonates, CRP was positive in $89\mathrm{\%}$ of the cases and was negative in $11\mathrm{\%}$ of cases.

Of the total cases, only $18\mathrm{\%}$ of the cases had culture-proven sepsis, $29\mathrm{\%}$ of cases had Probable sepsis, and $53\mathrm{\%}$ of the cases had suspected sepsis.

Pearson chi square 2.448; $P$ value 0.294(No significance)

The association between gender and sepsis was statistically not significant (P-value 0.294).

Pearson chi-square 9.89; $P$ value 0.007(Significant) The association between Gestational Age and sepsis was statistically significant (P-value 0.007).

In the above table, lower bound values were lowest in the Culture Proven Sepsis and higher bound values were observed in Suspected Sepsis. Thus Leucopenia, an adverse marker of sepsis, was observed in the study had differentiated the Culture Proven Sepsis and Suspected Sepsis. This difference reached levels of the highest statistical significance $(p=0.0001)$.

In the above table, lower bound values were lowest in the Culture Proven Sepsis and higher bound values were observed in Suspected Sepsis. Thus thrombocytopenia was a significant marker in diagnosing proven and probable sepsis. This trend reached levels of the highest statistical significance (p <0.0001).

In the above table, lower bound values were lowest in the Suspected Sepsis and higher bound values were found in Culture Proven Sepsis. Thus raised

CRP levels was a significant marker in diagnosing proven and probable Sepsis. This trend reached levels of the highest statistical significance (p < 0.0001).

In the above table, abnormal total leucocyte count was observed in $66.7\mathrm{\%}$ of the culture-positive sepsis and $24.4\mathrm{\%}$ of culture-negative cases. This abnormal trend of total leucocyte count in Proven Sepsis showed highly statistical significance (P-value 0.0001).

The total leucocyte count of this study had a moderate Negative predictive value (62.5%) with low

Sensitivity (33.3%), low Specificity (24.4%), and low Positive predictive value (8.8%).

In this study, thrombocytopenia occurred in $72.2\mathrm{\%}$ of the culture-positive sepsis, and $37.8\mathrm{\%}$ of culture-negative cases. This increased trend of thrombocytopenia in Proven Sepsis showed statistical significance (P-value 0.008).

In this study, Platelets had a high negative predictive value (91.1%), and moderate Sensitivity

(72.2%) with moderate Specificity (62.2%), and a low positive predictive value (29.5%).

In the above table, increased CRP levels was observed in $83.3\mathrm{\%}$ of the culture-positive sepsis and $90.2\mathrm{\%}$ of culture-negative cases. This association between CRP levels and culture did not show statistical significance (P-value 0.396).

In this study, CRP levels had high Sensitivity $(83.3\mathrm{\%})$, moderate Negative predictive value $(72.7\mathrm{\%})$ with low Specificity $(9.8\mathrm{\%})$, and low positive predictive value $(16.9\mathrm{\%})$.

IV. DISCUSSION

In this study, males were more (63%) compared to females (37%) which showed a male preponderance with a male to female ratio of 1.7:1. Similar male preponderance was reported by studies done by Bhalodia MJet al.8 (66.7%), Vinay BS et al.9(66.6%), Emad A. Morad et al.10 (66%), Abebe Sorsa11 (65.3%), Mittal A et al.12 (58.8%), Arnab Sengupta et al.13 (58.2%), Flora Chacha et al.14 (51.2%).

In the present study, low birth weight was reported in the majority (71%) of the study population. Similar reports were obtained in the study by Vinay BS et al.9, Mittal Aet al.12, Arnab Sengupta et al.13, Emad A. Morad et al.10. Contrast findings were seen in Flora Chacha et al.14, Abebe Sorsa study11.

In this study, more (62%) preterm babies were seen than term babies (38%). Similar to this finding preterm babies were more in Vinay BS et al.9 (68.4%), Choudhary D.K et al.15(76%) studies, whereas preterm babies were less in the study by Flora Chacha et al.14 (22.6%), Abebe Sorsa study11 (22.9%), Bhalodia MJet al.8 (26.7%), Emad A. Morad et al.10 (38%).

Of the total babies, $57\mathrm{\%}$ of the neonates were delivered by caesarean section $(57\mathrm{\%})$ and $43\mathrm{\%}$ were delivered by normal vaginal delivery (NVD). Whereas study by Flora Chacha et al. ${}^{14}$ $(22\mathrm{\%})$, Abebe Sorsa study ${}^{11}$ $(24.1\mathrm{\%})$, and Emad A. Morad et al. ${}^{10}$ $(46\mathrm{\%})$ caesarean sections was less compared to normal deliveries.

Early-onset of neonatal sepsis was observed among $56\mathrm{\%}$ of the neonates, and late-onset of neonatal sepsis was observed among $44\mathrm{\%}$ of neonates. Similar to this finding in Abebe Sorsa study[11], early onset of neonatal sepsis was seen in $61.2\mathrm{\%}$ of cases, and in $38.8\mathrm{\%}$ of cases late-onset of neonatal sepsis was reported, and Vinay BS et al.[38] also reported early-onset sepsis in $90\mathrm{\%}$ of cases.

In contrast to this study finding in the study by Choudhary D.K et al. ${}^{15}$ early-onset sepsis was present in $27\mathrm{\%}$ of cases, while late-onset neonatal sepsis was present in $73\mathrm{\%}$ of cases.

In this study, Blood culture was positive in only $18\mathrm{\%}$ of the cases and was negative in $82\mathrm{\%}$ of cases. Similar findings were reported by Choudhary D.K et al. ${}^{15}$ $(17\mathrm{\%})$, Harshitha M. Swamy et al. ${}^{16}$ $(20\mathrm{\%})$, Flora Chacha et al. ${}^{14}$ $(20.3\mathrm{\%})$, Bhalodia MJ et al. ${}^8$ $(38\mathrm{\%})$, whereas the higher incidence of positive blood culture was reported in the studies by Sriram R. ${}^{17}$ $(50.4\mathrm{\%})$, Vinay BS et al. ${}^9$ (80%).

In this study regarding culture, Staphylococcal species were present in $7\mathrm{\%}$ of the cases, followed by E.coli $(4\mathrm{\%})$, Klebsiella $(3\mathrm{\%})$, whereas Actinobacter species, Haemophilus, Methicillin-resistant staphylococcal aureus, and Pseudomonas aeruginosa was observed in each one percent of the cases. Staphylococcal species were the predominant organism. Similar to this study, S. aureus was the commonest organism in the studies by Karthikeyan G et al. ${}^{18}$, Misquith R et al. ${}^{19}$, Jaswal RS et al. ${}^{20}$, Tushar Priyanka et al. ${}^{21}$

CRP was positive in $89\mathrm{\%}$ and was negative in $11\mathrm{\%}$ of cases. Similar to this finding in Sriram. R study ${}^{17}$, CRP was positive in $88.7\mathrm{\%}$ cases and negative in $11.3\mathrm{\%}$ cases. In contrast to this finding Gurpreet Singh Chhabra et al. ${}^{22}$ studies had $3\mathrm{\%}$ CRP positivity.

a) Total WBC Count vs Blood Culture Positivity

In this study, abnormal total leucocyte count was observed in $66.7\mathrm{\%}$ of the culture-positive sepsis and $24.4\mathrm{\%}$ of the culture-negative cases. The total leucocyte count of this study had reported $33.3\mathrm{\%}$ Sensitivity, which was in accordance with studies by Tushar Priyanka et al.[21] $(23.63\mathrm{\%})$, Harshitha M. Swamy et al.16, and Makkar M et al.[23] $(43.18\mathrm{\%})$.

In this study total leucocyte count had shown specificity of $24.4\mathrm{\%}$, which was lower than other studies by Tushar Priyanka et al.[21] $(71.27\mathrm{\%})$, Bhalodia et al.[8] $(74.5\mathrm{\%})$, and Majumdar A et al.[24] $(85\mathrm{\%})$.

This variation might be due to different selection criteria adapted while selecting the participants, and different levels of infections in neonates.

In this study total leucocyte count had a low positive predictive value (8.8%), which was similar to the study by Hiral PS ${}^{25}(15.38\mathrm{\%})$. Contrast results were obtained in the studies by Punyashetty KB et al. ${}^{26}$ (87.5%), Makkar M et al. ${}^{23}$ (86.36%), and Narasimha A et al. ${}^{27}(80\mathrm{\%})$.

The total leucocyte count of this study had a moderate Negative predictive value (62.5%), which was in accordance with Makkar M et al. 23, Majumdar A et al. 24, and Bhalodia et al. 8 studies.

b) Platelet Count vs Blood Culture Positivity

This study had high Sensitivity (72.2%) for platelets which was in accordance with Makkar M et al.23, Majumdar A et al.24, Hiral PS 25, Mittal Aet al.12.

For platelets, moderate Specificity (62.2%) was observed in this study which was in line with studies by Bhalodia et al.8, Hiral PS25, and Narasimha A et al.27. Low Positive predictive value (29.5%) was reported which was in concordance with Khair BK et al.28, Hiral PS25, Mittal Aet al.12, and Harshitha M. Swamy et al.16.

In this study, Platelets had a high negative predictive value (91.1%), which was similar to studies of Hiral PS ${}^{25}(89.58\mathrm{\%})$, Punyashetty KB et al. ${}^{26}$ (93.5%), Khair BK et al. ${}^{28}$ (94%), Majumdar A et al. ${}^{24}$ (95%), and Narasimha A et al. ${}^{27}$ (85.71%)

c) CRP vs Blood Culture Positivity

In this study in $83.3\mathrm{\%}$ of the culture-positive sepsis and $90.2\mathrm{\%}$ of culture-negative cases increased CRP levels were reported. This increased CRP levels in culture-proven sepsis did not show statistical significance (P-value 0.396).

In this study, CRP levels had high sensitivity $(83.3\mathrm{\%})$, which was similar to Chandra A et al.29, Patel U et al.[30] $(81.7\mathrm{\%})$, Vinay BS et al. study[9] $(81.2\mathrm{\%})$, Sharma CM et al.[31] $(80\mathrm{\%})$, Harshitha M. Swamy et al.[16] $(90\mathrm{\%})$.

CRP levels in this study had low specificity $(9.8\mathrm{\%})$, which was less than studies of Chandra A et al.[29] $(42\mathrm{\%})$, Harshitha M. Swamy et al.[16] $(47.5\mathrm{\%})$. This variation could be because of the different methodologies used to measure CRP and the cut off used.

CRP levels in this study had low PPV (16.9%), which was similar to studies of Flora Chacha et al. ${}^{14}$ (37.5%), Harshitha M. Swamy et al. ${}^{16}$ (30%).

In this study, CRP levels had moderate NPV (72.7%), which was similar to studies of Sucilathangam G et al.32 (78.1), Flora Chacha et al.14 (84.5%), Harshitha M. Swamy et al.16 (95%).

In conclusion, it is proved that no single individual test is better than others in detecting neonatal sepsis. So, the conjunction of tests like Total WBC, Platelets and CRP can be utilized for better sepsis screening, timely management and to reduce the duration of hospital stay and to improve appropriate antibiotic utilization.

ACKNOWLEDGEMENT

None

Author's Contribution:

Vamseekrishna Polepalli: Definition of intellectual content, Literature survey, Prepared first draft of manuscript, implementation of study protocol, data collection, data analysis, manuscript preparation and submission of article;

Rohini Reddy Vanukuri: Concept, design, clinical protocol, manuscript preparation, editing, and manuscript revision;

Nayan Baba Pelala: Design of study, statistical Analysis and Interpretation;

Conflict of Interest: No! Conflict of interest is found elsewhere considering this work.

Source of Funding: There was no financial support concerning this work.