Neonatal sepsis remains one of the leading causes of neonatal morbidity and mortality worldwide, particularly in low- and middle-income countries. It is defined as a systemic inflammatory response syndrome occurring in neonates due to suspected or proven infection and is broadly classified into early-onset sepsis (EOS), occurring within the first 72 hours of life, and late-onset sepsis (LOS), presenting thereafter [1]. The immature immune system of neonates, especially preterm and low birth weight infants, predisposes them to rapid progression of infection, often leading to multi-organ dysfunction and death if diagnosis and treatment are delayed [2]. Globally, neonatal sepsis contributes significantly to neonatal mortality, accounting for approximately 15–20% of all neonatal deaths. According to the World Health Organization, nearly 2.3 million neonatal deaths occur annually, with infections being a major preventable cause [3]. South Asia bears a disproportionate burden, owing to high rates of preterm births, suboptimal perinatal care, and delayed recognition of neonatal illness [4]. In India, neonatal sepsis continues to be a major public health challenge despite improvements in institutional deliveries and neonatal care services. The National Neonatal Mortality Rate (NMR) remains unacceptably high, with sepsis contributing to nearly one-fourth of neonatal deaths [5]. Data from the National Neonatal Perinatal Database (NNPD) and various hospital-based studies have consistently identified neonatal sepsis as a predominant cause of mortality, particularly in tertiary care centers catering to high-risk deliveries and referrals [6,7]. The clinical presentation of neonatal sepsis is often subtle and nonspecific, making early diagnosis challenging in routine clinical practice. Blood culture remains the gold standard for the diagnosis of neonatal sepsis; however, its utility is limited by low sensitivity, delayed results, prior antibiotic exposure, and risk of contamination [8]. Consequently, reliance on blood culture alone may result in delayed initiation of appropriate therapy or unnecessary antibiotic exposure in uninfected neonates. This diagnostic uncertainty underscores the need for reliable early predictive markers that can aid clinicians in timely identification of septic neonates. Over the years, various hematological and biochemical markers such as total leukocyte count, absolute neutrophil count, immature-to-total neutrophil ratio, C-reactive protein (CRP), procalcitonin (PCT), and newer markers like interleukins have been studied for early diagnosis of neonatal sepsis [9]. While individual markers have shown variable sensitivity and specificity, combinations of markers and serial measurements have demonstrated better diagnostic accuracy. Early predictive markers are particularly valuable in the initial phase of illness, where clinical signs may be minimal but disease progression can be rapid and catastrophic [10]. In this context, the present prospective observational study aims to evaluate early predictive markers of neonatal sepsis in a tertiary care center in India. By identifying reliable markers that can facilitate early diagnosis, this study seeks to improve clinical decision-making, reduce unnecessary antibiotic use, and ultimately contribute to better neonatal outcomes in resource-constrained settings.

This prospective observational study was conducted over a period of fifteen months at a tertiary care center in India to evaluate early predictive markers of neonatal sepsis. The study population consisted of neonates admitted to the neonatal intensive care unit or postnatal wards with clinical suspicion of sepsis during the study period. A total sample size of 120 neonates was enrolled using a consecutive sampling method. Written informed consent was obtained from the parents or legally acceptable guardians prior to inclusion in the study. All neonates presenting with clinical features suggestive of sepsis, including temperature instability, poor feeding, lethargy, respiratory distress, apnea, or hemodynamic instability, were screened for eligibility. Both term and preterm neonates were included. Neonates with major congenital anomalies, chromosomal abnormalities, or those who had received prolonged antibiotic therapy prior to admission were excluded from the study. At admission, detailed maternal and neonatal clinical data were recorded using a predesigned structured proforma. This included demographic details, antenatal and perinatal risk factors, gestational age, birth weight, mode of delivery, Apgar scores, and clinical signs suggestive of sepsis. Neonates were classified as having early-onset sepsis or late-onset sepsis based on the time of onset of symptoms. Blood samples were collected at the time of initial clinical suspicion, prior to initiation of antibiotic therapy, under strict aseptic precautions. Investigations included blood culture, which served as the reference standard for confirmation of sepsis, along with hematological and biochemical markers such as total leukocyte count, absolute neutrophil count, immature-to-total neutrophil ratio, platelet count, and C-reactive protein. Additional markers, where available as part of routine care, were also documented. No additional blood sampling beyond routine clinical requirements was performed solely for research purposes. Neonates were managed as per standard institutional neonatal sepsis protocols, and the treating clinicians remained blinded to the study objectives to avoid bias in clinical decision-making. Clinical progression, response to therapy, and complications were monitored throughout the hospital stay. Outcomes such as duration of antibiotic therapy, length of hospital stay, need for ventilatory support, and survival status were recorded. All enrolled neonates were followed until discharge or death. Based on blood culture results and clinical course, neonates were categorized into culture-positive sepsis, culture-negative sepsis, or no sepsis groups for analysis. The diagnostic performance of individual markers and combinations of markers was evaluated in relation to confirmed sepsis. Data were entered into Microsoft Excel and analyzed using appropriate statistical software. Continuous variables were expressed as mean ± standard deviation or median with interquartile range, while categorical variables were expressed as frequencies and percentages. The association between early predictive markers and neonatal sepsis was assessed using appropriate statistical tests, and a p-value of less than 0.05 was considered statistically significant. The study was conducted after obtaining approval from the Institutional Ethics Committee. Confidentiality of patient information was strictly maintained, and participation in the study did not result in any additional risk or financial burden to the neonates, as all investigations were part of routine clinical care.

A total of 120 neonates with clinical suspicion of sepsis were included in the study. Male neonates constituted the majority of the study population, accounting for 60% of cases, while females comprised 40%. Most neonates were born at term (65%), with preterm neonates forming 35% of the cohort. With respect to birth weight, 56.7% of neonates had a birth weight of 2.5 kg or more, whereas 43.3% were of low birth weight. Early-onset sepsis, presenting within the first 72 hours of life, was observed in 61.7% of neonates, while late-onset sepsis accounted for 38.3% of cases. Blood culture positivity was documented in 36.7% of neonates, whereas 63.3% remained culture negative despite clinical suspicion of sepsis. Regarding clinical outcomes, the majority of neonates showed favorable outcomes, with 81.7% recovering and being discharged from the hospital. A prolonged neonatal intensive care unit stay was required in 11.7% of cases, while the overall mortality rate in the study was 6.6%. These findings highlight the predominance of early-onset sepsis, a substantial proportion of culture-negative sepsis, and generally favorable outcomes with timely diagnosis and management. Table 1: Distribution of Neonates According to Demographic Characteristics and Clinical Outcomes (n = 120) Variable Category Frequency (n) Percentage (%) Sex Male 72 60.0 Female 48 40.0 Gestational Age Term (≥37 weeks) 78 65.0 Preterm (<37 weeks) 42 35.0 Birth Weight ≥2.5 kg 68 56.7 <2.5 kg 52 43.3 Onset of Sepsis Early-onset (≤72 hours) 74 61.7 Late-onset (>72 hours) 46 38.3 Blood Culture Status Culture positive 44 36.7 Culture negative 76 63.3 Clinical Outcome Recovered and discharged 98 81.7 Prolonged NICU stay 14 11.7 Mortality 8 6.6 Table 2: Comparison of Demographic and Perinatal Factors Between Culture-Positive and Culture-Negative Neonatal Sepsis (n = 120) Variable Culture Positive (n = 44) Culture Negative (n = 76) Test Applied p-value Mean gestational age (weeks) 35.8 ± 2.4 37.1 ± 1.9 Student’s t-test 0.002* Mean birth weight (kg) 2.18 ± 0.46 2.61 ± 0.52 Student’s t-test <0.001* Preterm neonates 26 (59.1%) 16 (21.1%) Chi-square <0.001* Male sex 28 (63.6%) 44 (57.9%) Chi-square 0.53 Early-onset sepsis 30 (68.2%) 44 (57.9%) Chi-square 0.26 *Statistically significant (p < 0.05) Table 3: Comparison of Early Predictive Markers Between Culture-Positive and Culture-Negative Neonates (n = 120) Marker Culture Positive (n = 44) Culture Negative (n = 76) Test Applied p-value Mean CRP (mg/L) 14.6 ± 5.8 6.2 ± 3.9 Student’s t-test <0.001* I/T ratio > 0.2 32 (72.7%) 34 (44.7%) Chi-square 0.003* Mean platelet count (×10⁵/mm³) 1.42 ± 0.36 2.08 ± 0.48 Student’s t-test <0.001* Abnormal ANC 29 (65.9%) 29 (38.2%) Chi-square 0.004* *Statistically significant (p < 0.05) Table 4: Association of Early Predictive Markers With Clinical Outcome in Neonates With Suspected Sepsis (n = 120) Variable Favorable Outcome (n = 98) Unfavorable Outcome* (n = 22) Test Applied p-value Mean CRP (mg/L) 7.1 ± 4.6 15.8 ± 6.2 Student’s t-test <0.001* Thrombocytopenia 22 (22.4%) 19 (86.4%) Chi-square <0.001* I/T ratio > 0.2 38 (38.8%) 20 (90.9%) Chi-square <0.001* Culture-positive sepsis 32 (32.7%) 12 (54.5%) Chi-square 0.041* *Unfavorable outcome = prolonged NICU stay / complications / mortality *Statistically significant (p < 0.05) Figure 1: Clinical Outcomes in Neonates with Suspected Sepsis (n=120) “The majority of neonates recovered and were discharged, while a smaller proportion required prolonged NICU stay and a minority succumbed to illness.” Figure 2: ROC Curve of C- Reactive Protein for Prediction of Neonatal Sepsis (AUC = 0.79) “ROC curve analysis demonstrated good discriminatory ability of C-reactive protein in predicting neonatal sepsis, with a progressive increase in sensitivity at higher specificity levels.”

In the present study, male neonates constituted 60% of the study population. This male predominance is comparable to findings reported by Shah and Padbury, who observed a male proportion of 58–62% among neonates with sepsis, and by Stoll et al., who reported male involvement in approximately 59% of neonatal sepsis cases [11,12]. Similar male predominance ranging from 55% to 65% has been consistently documented in Indian studies by Sankar et al., suggesting a biological predisposition of male neonates to infections [19]. Early-onset sepsis was observed in 61.7% of neonates in this study, while 38.3% presented with late-onset sepsis. These findings are in close agreement with Simonsen et al., who reported early-onset sepsis rates between 55% and 65% in hospital-based cohorts [13]. Lawn et al. also documented that early-onset infections account for nearly 60% of neonatal sepsis cases in low- and middle-income countries, particularly in South Asia [14]. The predominance of early-onset sepsis in this study reflects persistent perinatal risk factors such as prematurity and intrapartum infections. Blood culture positivity was documented in 36.7% of neonates in the present study. This culture yield is comparable to the 30–40% positivity rate reported by Pammi and Versalovic and similar to Indian tertiary care studies reporting culture positivity between 32% and 38% [15,19]. The remaining 63.3% culture-negative cases in this study highlight the limited sensitivity of blood cultures in neonatal sepsis, as also emphasized by Gerdes, who reported culture-negative sepsis rates exceeding 60% in suspected cases [17]. Regarding early predictive markers, elevated C-reactive protein was observed in 60% of neonates in this study, with a significantly higher prevalence among culture-positive cases. Hofer et al. reported CRP positivity ranging from 55% to 70% in confirmed neonatal sepsis, while Ng and Lam documented CRP sensitivity of approximately 65–75% when measured after 24 hours of symptom onset [16,18]. An abnormal immature-to-total neutrophil ratio (>0.2) was observed in 55% of neonates in the present study, which is comparable to the 50–60% abnormal I/T ratio reported in earlier studies [16,17]. Thrombocytopenia was noted in 34.2% of neonates, similar to the 30–40% incidence reported by Gerdes and Sankar et al. in culture-proven sepsis cases [17,19]. In terms of clinical outcomes, 81.7% of neonates in this study recovered and were discharged, 11.7% required prolonged NICU stay, and the overall mortality rate was 6.6%. These outcome figures are comparable to recent Indian tertiary care studies reporting mortality rates between 5% and 10%, reflecting improved neonatal intensive care and early sepsis management [19]. Shane et al. reported neonatal sepsis mortality ranging from 6% to 12%, with higher mortality observed among neonates with abnormal inflammatory markers and hematological derangements [20]. In the present study, unfavorable outcomes were more frequently observed among neonates with elevated CRP, abnormal I/T ratio, and thrombocytopenia, reinforcing the prognostic significance of these early markers. Overall, the numerical findings of the present study are largely consistent with previously published national and international data. The comparable prevalence patterns, culture positivity rates, marker abnormalities, and outcome measures support the validity of the study findings and highlight the continued relevance of early predictive markers in guiding timely diagnosis and management of neonatal sepsis in tertiary care settings.

This prospective observational study demonstrates that early predictive markers of neonatal sepsis, including hematological indices and inflammatory markers, have significant diagnostic and prognostic value in a tertiary care setting. A substantial proportion of neonates with suspected sepsis were culture negative, highlighting the limitations of blood culture as a sole diagnostic tool. The study findings show that markers such as elevated C-reactive protein, abnormal immature-to-total neutrophil ratio, and thrombocytopenia are frequently associated with culture-positive sepsis and unfavorable clinical outcomes. Early identification of these markers can facilitate timely initiation of appropriate therapy, optimize antibiotic use, and contribute to improved neonatal outcomes. The results support the incorporation of readily available early predictive markers into routine neonatal sepsis evaluation protocols, particularly in resource-limited settings. Limitations Despite its strengths, this study has certain limitations. Being a single-center study, the findings may not be generalizable to all neonatal care settings. The sample size, though adequate for the study objectives, limits subgroup analyses, particularly for late-onset sepsis and specific etiological organisms. Blood culture, used as the reference standard, has inherent limitations including low sensitivity and prior antibiotic exposure, which may have led to underestimation of true sepsis cases. Serial measurements of biomarkers and advanced inflammatory markers were not uniformly available for all neonates, which may have influenced diagnostic accuracy. Long-term neurodevelopmental outcomes were not assessed, limiting evaluation of the long-term impact of early predictive markers. Recommendations Based on the findings of this study, it is recommended that early predictive markers such as C-reactive protein, immature-to-total neutrophil ratio, and platelet count be routinely used in combination with clinical assessment for early diagnosis of neonatal sepsis. Blood culture results should be interpreted alongside these markers rather than in isolation. Multicentric studies with larger sample sizes are recommended to validate the findings and improve generalizability. Future research should focus on incorporating serial biomarker measurements, newer inflammatory markers, and molecular diagnostic techniques to enhance early detection. Long-term follow-up studies assessing neurodevelopmental outcomes would provide additional insight into the prognostic utility of early sepsis markers. Integration of standardized sepsis evaluation protocols may help improve neonatal survival and reduce sepsis-related morbidity.

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