When pathogenic organisms invade the bloodstream, they can trigger severe infections such as septicaemia, and when localized to specific sites like the lungs or meninges, they may cause pneumonia or meningitis. A systemic inflammatory response is defined by the presence of at least two of the following: fever or hypothermia, tachycardia, tachypnoea, or abnormal white cell counts with immature forms.[1] This condition contributes to 30–50% of neonatal deaths in developing countries.[2] Neonatal sepsis is categorized as early-onset if it occurs within the first 72 hours and late-onset if it occurs thereafter.[3,4] Early-onset sepsis typically stems from pathogens in the maternal genital tract or hospital environment, while late-onset sepsis is usually nosocomial or community-acquired.[5,6] Various biomarkers—including acute-phase proteins, cytokines, surface antigens, and bacterial genomes—have been explored for early diagnosis, but many are costly or require advanced technology, limiting their use in resource-limited settings.[7] An ideal diagnostic test must be rapid, affordable, and possess high sensitivity, specificity, and predictive values to effectively rule out sepsis and prevent unnecessary antibiotic use. C-reactive protein (CRP), first described by Tillet and Francis in 1930, binds complement to foreign or damaged cells and peaks approximately 50 hours after an inflammatory trigger.[8] Although nonspecific, CRP is a valuable adjunct to clinical findings in diagnosing sepsis.[9] Hematologic parameters such as red cell distribution width (RDW), leukocyte count, mean platelet volume, and platelet distribution width (PDW) are routinely measured in complete blood counts. RDW reflects erythrocyte size variation and is commonly used in anaemia assessment; it has also been linked to conditions like chronic urticaria, gram-negative bacteraemia, and is considered prognostic in diseases such as stable angina.[10] Zahorec R[11] proposed the neutrophil-lymphocyte ratio (NLR) as an infection marker, and later research by Loonen AJ et al[12] found NLR to be a promising and rapidly available biomarker for distinguishing bloodstream infections. NLCR may outperform traditional markers such as CRP, leukocyte, and neutrophil counts in predicting bacteraemia.[13] This study therefore investigated RDW and NLR as potential diagnostic markers for neonatal sepsis and their correlation with CRP values.

This cross-sectional study was conducted in the NICU (neonatal intensive care unit) of a tertiary care hospital between January 2021 and July 2022, following approval from the Institutional Ethics Committee. The study group consisted of 80 neonates presenting with clinical features of sepsis—either culture-positive or with positive sepsis screening and/or high-risk factors. An equal number of age- and gender-matched neonates without signs of sepsis, negative sepsis screening, and no identifiable risk factors served as controls. Neonates with major congenital anomalies, prior antibiotic exposure, or lack of parental consent were excluded. Informed written consent was obtained from all parents or legal guardians prior to their inclusion in the study. A uniform protocol guided the assessment of all participants. Data collection included clinical history, physical examination, and relevant investigations documented using a structured proforma. Maternal data such as gestational age, obstetric history, and antenatal complications were also recorded. Prior to initiating antibiotics, all neonates underwent CBC (including MPV, PDW, and RDW) and CRP testing; blood cultures were obtained only in suspected sepsis cases. CBC samples were processed via the CELLTAC ES analyzer using EDTA tubes, with results available within five minutes. CRP was analyzed by mixing 50 µL of centrifuged serum with one drop of CRP antigen. For blood cultures, 2 mL of venous blood was aseptically inoculated into culture bottles and incubated at 37°C for 5–7 days; positive samples were subcultured and organisms identified using standard methods. Treatment for sepsis followed institutional protocols.

Statistical analysis

Data were entered in MS Excel 2007 and analyzed using SPSS version 16. Normality of data was assessed using the Kolmogorov–Smirnov test. Between-group comparisons for normally distributed variables were performed using the unpaired t-test, while categorical variables were analyzed using the chi-square test. Spearman’s correlation was used to evaluate the relationship between CRP and hematological parameters. A p-value < 0.05 was considered statistically significant.

In this study, 80 patients were included in both the case and control groups. Of the 160 neonates included in this study, 80 were male and 80 were female, maintaining a male-to-female ratio of 1:1 in both the groups. Mode of delivery was delivery was lower segment cesarean section (LSCS) in 92 neonates and vaginal in 68 neonates.  Most neonates were delivered at full term in both the case (66.2%) and control groups (91.2%). Majority of neonates were born to multigravida (55%) in both groups. The mean birth weight of neonates in case group was significantly lower than control group (p<0.05). (Table 1).

Table 1: Characteristics of Study Participants

Meconium aspiration and premature rupture of membranes (PROM), observed in 13 neonates each, were identified as the most common risk factors for sepsis in this study. The most frequently observed clinical manifestation was respiratory distress which was observed in 56 neonates. (Table 2).

Table 2: Predisposing factors leading to Sepsis in Study Participants

There was statistically significant difference in RDW and NLR between case and control groups (p<0.001). (Table 3)

Table 3: Mean and Standard Deviation of Study Parameters in Cases and Controls

RDW was more than 16 (>16) in 55 cases and 36 controls, and this difference amongst the two groups was statistically significant (p value = 0.002). NLR was more than 2 (>2) in 65 cases and 22 controls, suggesting significant association between NLR and neonates with sepsis (p <0.001). (Table 4)

Table 4: RDW and NLR in Cases and Controls

RDW >16 had a sensitivity of 68.8% and specificity of 55%, while NLR >2 had a sensitivity of 81.35% and specificity of 72.5% for the diagnosis of neonatal sepsis. (Table 5)

Table 5: Validity of RDW and NLR as a Marker in Neonatal Sepsis

Neonatal sepsis, defined as a systemic inflammatory response or bacterial isolation from blood within the first 28 days, remains a leading cause of neonatal admissions and contributes to 26% of global neonatal deaths.[1,14] In developing nations, it accounts for 30–50% of such deaths.[2] Although blood culture is the diagnostic gold standard, it is time-intensive (48–72 hours) and only detects pathogens in 10–60% of cases.[15] Therefore, there is a need for a rapid, affordable screening method for early identification. Limited research in India has explored the diagnostic utility of red cell parameters in neonatal sepsis.[16] This study aimed to evaluate the diagnostic potential of RDW and NLR in neonatal sepsis and examine their correlation with CRP levels.

We observed no significant difference in gender distribution between neonates with sepsis and healthy neonates in our study. These findings are in agreement with previous studies.[17,18] However, Shah GS et al [19] and Mehar V et al[20] have observed male predominance among neonates with sepsis in their respective studies. In our study, sepsis was more commonly observed in neonates delivered by LSCS (60%).  Similarly, caesarean delivery was associated with increased odds of neonatal sepsis as compared with vaginal delivery in a previous.[21] In our study, a significantly higher proportion of neonates with sepsis were born to multigravida mothers (58.8%) compared to primigravida (41.2%). Similarly, one study reported more normal vaginal deliveries in the control group, while LSCS was more common in the sepsis group (p<0.001).[22] Tewabe T et al [23] also found that over half (55.1%) of the mothers in their Ethiopian NICU study were multigravida.

Premature neonates are at a higher risk of infection mainly due to deficient immune system, mainly due to decreased IgG antibodies and incompetent opsonization and complement activation. In our study, 53 of the 80 neonates with sepsis were born at term, while in the control group, 73 were term and only 7 were preterm (p<0.001). Shah GS et al[19], in a similar study, observed that prematurity increased the risk of sepsis by 4.85 times (p < 0.001). A systematic review confirmed early gestational age as a significant risk factor for neonatal sepsis.[24] In a cross-sectional study by Bangi VAB et al[25], 61 out of 120 neonates with sepsis were preterm, showing a significantly higher sepsis rate in preterms (65.2%) than in term neonates (21.6%). Similarly, another study found that preterm neonates had a 1.49 times higher risk of developing septicemia compared to term neonates (p<0.05).[20] In our study, the mean birth weight was lower in sepsis group (2.5 kg) compared to controls (2.8 kg) (p < 0.05). Similar observations were made in previous studies.[19,24]

The maternal and neonatal risk factors were assessed in neonates with sepsis in our study. We observed that in the neonates who had sepsis, 16.2% mothers had MSAF, 16.2% had PROM, 2.5% mothers had fever and 6.2% mothers had UTI (Urinary tract infection) at the time of delivery. (Table 2) Oddie S et al[26], in a similar study, observed that PROM (odds ratio 13.7, 95% confidence interval 4.8 to 39.5) and prelabour rupture of the membranes (odds ratio 10.7, 95% confidence interval 3.8 to 30.1) were independent and statistically significant risk factor for infection. Another study found that maternal factors having a significant risk for the development of sepsis were PROM, MSAF and foul smelling liquor.[19] The authors found that risk of sepsis in PROM and MSAF was about 2 times higher as compared to the control group. Similar observations were made by Murthy S et al[24].

In our study, the most common clinical signs among neonates with sepsis were respiratory distress (70%), followed by lethargy (16.5%), feeding refusal (5%), and hypotension (2.5%) (Table 2). In a similar study, Fanaroff AA et al[27] found that apnea (55%), gastrointestinal signs like feeding intolerance and abdominal distension (43%), increased respiratory support (29%), and lethargy or hypotonia (23%) were key indicators.

Lim WH et al[28] reported poor activity (48.7%) and increased respiratory effort (43%) as the most frequent symptoms. Similarly, Stoll BJ et al[29] noted that respiratory and cardiovascular manifestations were most common in neonatal sepsis.

RDW indicates variation in red blood cell size and is influenced by infection and inflammation. It has been proposed as a potential marker for adverse outcomes in sepsis.[17] In our study, RDW values above 17% were significantly associated with neonatal sepsis, showing 68.8% sensitivity, 55% specificity, 60.4% PPV, and 63.8% NPV. Although a weak positive correlation with CRP was observed, it was not statistically significant (p > 0.05). Other studies have also reported similar findings. RDW cut-off values around 17.25–17.9% demonstrated good sensitivity (83–86%) and specificity (50–87%) for detecting sepsis.[17,30] In large cohorts, RDW fluctuations showed greater diagnostic accuracy than static values, with AUCs up to 0.88 and sensitivity above 90%.[31] Higher RDW levels were also significantly correlated with CRP in septic neonates, reinforcing its diagnostic potential.[32,33] Additionally, RDW levels in sepsis cases were consistently higher than in healthy controls, supporting its utility as a supplementary marker.[31]

In our study, NLR>2 showed a sensitivity of 81.3% and specificity of 72.5% for diagnosing neonatal sepsis. The positive predictive value (PPV) was 74.7%, and negative predictive value (NPV) was 79.5%, suggesting NLR as a reliable marker. A weak positive, non-significant correlation with CRP was also noted (p>0.05). Similar correlations between NLR and CRP in neonatal sepsis have been observed in previous research.[34,35] Other studies reported NLR cut-offs between 1.4 and 2.7 with varying diagnostic performance. Sensitivity ranged from 68.3% to 88%, while specificity varied between 42.3% and 84%.[34-37] One study found that combining NLR with CRP improved diagnostic accuracy to over 70%.[34]. Another reported higher mean NLR in septic neonates versus controls, with significant ROC curve results supporting its diagnostic value.[35] Additional findings highlighted NLR as an independent predictor of neonatal sepsis even after adjusting for clinical variables, and its diagnostic potential remained independent of other markers like PCT and CRP. There were few limitations in our study including smaller sample size and single center study.

PROM, meconium aspiration, parity, prematurity, and low birth weight were identified as risk factors for neonatal sepsis. RDW and NLR were significantly elevated in septic neonates, with NLR showing the highest diagnostic efficiency. However, none showed significant correlation with CRP. These simple, cost-effective, and routinely available parameters could serve as useful diagnostic tools for neonatal sepsis, especially in resource-limited settings.

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