Neonatal sepsis is a clinical syndrome characterized by bacteremia accompanied by systemic signs and symptoms of infection occurring within the first month of life. It encompasses a wide spectrum of systemic infections in newborns, including septicemia, meningitis, pneumonia, arthritis, osteomyelitis, and urinary tract infections¹. Globally, approximately 130 million live births occur annually, of which nearly 4 million neonatal deaths are reported within the first four weeks of life. The major direct causes of neonatal mortality include preterm birth (28%), severe infections (26%), and birth asphyxia (23%)². Data from the National Neonatal Perinatal Database (NNPD, 2002–03), collected from 18 centers across India, report an incidence of neonatal sepsis of 29.9 per 1,000 intramural live births. Early-onset sepsis accounts for nearly 67% of all neonatal sepsis cases, while meningitis contributes to 10.6% of these infections³. Neonatal sepsis remains a significant contributor to neonatal mortality, accounting for approximately 16% of intramural neonatal deaths³. Despite advances in antimicrobial therapy, neonatal sepsis continues to pose a major clinical challenge due to its high incidence, variable clinical presentation, and associated mortality. The signs and symptoms are often nonspecific, necessitating rapid identification of infected neonates to initiate timely treatment. Although blood culture remains the gold standard for diagnosing neonatal septicemia, it has several limitations, including low sensitivity, delayed results, requirement of specialized laboratory facilities, larger blood volumes, and a culture positivity rate of only about 40%. Furthermore, only 3–5 per 1,000 neonates admitted to neonatal intensive care units have culture-proven septicemia⁴. An ideal diagnostic test for neonatal sepsis should reliably differentiate infected from non-infected neonates, be cost-effective, technically simple, and demonstrate high sensitivity and specificity with minimal false-positive and false-negative results. In recent decades, considerable attention has been directed toward the role of acute-phase reactants in the diagnosis and monitoring of neonatal sepsis. These proteins increase in response to inflammation, with systemic infection being the primary trigger in neonates. Among the various acute-phase reactants, C-reactive protein (CRP) has emerged as a sensitive and reliable marker for neonatal sepsis. CRP has been shown to be superior to conventional indicators such as total leukocyte count, band cell-to-total neutrophil ratio, degenerative changes in neutrophils, buffy coat smear, platelet count, micro erythrocyte sedimentation rate, and gastric aspirate cytology. Quantitative estimation of serum CRP can be performed using immunonephelometry or ELISA within one hour, requiring less than 500 µL of blood. In settings where immunonephelometry or ELISA is unavailable, latex agglutination techniques offer qualitative or semi-quantitative assessment of CRP. These methods are rapid, simple to perform, cost-effective, and associated with minimal technical errors and inter-observer variability, with relatively low rates of false-positive and false-negative results. Aim of the study: 1. To evaluate the effect of intrapartum risk factors for early-onset sepsis (EOS) on CRP levels in neonates. 2. To assess the diagnostic utility of C-reactive protein in the early identification of neonatal sepsis.

Study Design and Setting This prospective observational study was conducted over one year (March 2022 to March 2023)in the Newborn Care Unit of ACSR Government Medical College Hospital, Nellore. A total of 108 neonates with identified intrapartum risk factors for early-onset sepsis (EOS) were enrolled after obtaining informed written parental consent. Sample Size Calculation The sample size was calculated based on the expected proportion of elevated CRP levels among neonates at risk of infection, using a confidence level of 95% and an absolute precision of 10%. Assuming the prevalence of neonatal infection among at-risk neonates to be 50% (as reported in previous studies and to yield the maximum sample size), the sample size was calculated using the formula: n=(Z^2×p×q)/d^2 Where: Z = 1.96 (for 95% confidence interval) p = 0.5 q = 1 − p = 0.5 d = 0.1 (absolute precision) n=(1.96)^2│0.5│0.5┤/(0.1)^2 ┤ =96 After accounting for an anticipated 10–12% attrition or non-response rate, the final sample size was rounded up to 108 neonates. Study Population Neonates born to mothers with at least one intrapartum risk factor—premature rupture of membranes, more than three vaginal examinations after membrane rupture, maternal fever within one week prior to delivery, foul-smelling liquor, prolonged labor, meconium-stained liquor, or untreated/partially treated maternal urinary tract infection—were included. Neonates with gestational age<28 weeks (extreme preterm), birth weight<1 kg (extreme low birth weight), congenital anomalies, or those born outside the institution were excluded. Clinical Evaluation Detailed maternal and perinatal history was recorded. Gestational age was assessed using the Modified Ballard Scoring System. Neonates were monitored clinically for 72 hours for signs suggestive of sepsis involving general, respiratory, gastrointestinal, cardiovascular, central nervous system, and hematological systems. Laboratory Investigations Cord blood samples were collected at birth for CRP estimation. Between 24 and 36 hours of life, venous blood samples were obtained for CRP, total leukocyte count (TLC), absolute neutrophil count (ANC), band cell count, and blood culture. Abnormal parameters: TLC (Total Leukocyte Count): A count of less than 5,000 cells/mm³ is defined as abnormal (specifically, leukopenia). ANC (Absolute Neutrophil Count): A count of less than 1,500 cells/mm³ is defined as abnormal (specifically, neutropenia). Band Cell Count: A count greater than 20% of all neutrophils is defined as abnormal (indicating a "left shift", often seen in acute infections or inflammation). Blood Culture Blood culture was performed by inoculating 1 mL of blood into Brain Heart Infusion broth and incubated aerobically at 37°C for seven days. Subcultures were done at 24 hours, 72 hours, and on day seven on standard culture media. Organisms were identified using standard microbiological techniques. Cultures with no growth after seven days were reported as negative. C-Reactive Protein Estimation Serum CRP was estimated using a rapid slide latex agglutination method employing qualitative and semi-quantitative techniques. CRP levels ≥0.6 mg/dL were considered positive. Classification of Sepsis Neonates were categorized as: Definite sepsis: Clinical signs with positive blood culture Probable sepsis: Clinical signs with abnormal laboratory parameters No sepsis: Absence of clinical and laboratory evidence Statistical Analysis Descriptive statistics were applied. Risk ratios were calculated for CRP levels in cord and neonatal blood. Fisher’s exact test was used to assess the association between CRP levels and sepsis. Sensitivity, specificity, positive predictive value, and negative predictive value of CRP were determined. Statistical analysis was performed using SPSS version 15.0 and other standard statistical

A total of 108 neonates with intrapartum risk factors for early-onset sepsis (EOS) were included. Of these, 64 (59.3%; 90% CI: 51.35–66.72) were male and 44 (40.7%; 90% CI: 33.28–48.25) were female. Low birth weight (<2.5 kg) was observed in 20 neonates (18.5%; 90% CI: 13.17–25.41), while 15 (13.9%; 90% CI: 9.29–20.25) were born at ≤37 weeks gestation. Most neonates were born to primiparous mothers (66.7%; 90% CI: 58.88–73.64). Vaginal delivery occurred in 63 cases (58.3%) and LSCS in 45 cases (41.7%). Baseline demographic and obstetric characteristics are summarized in Table 1. Regarding intrapartum risk factors, rupture of membranes (ROM) lasting 12–24 hours was most common (53.7%), followed by>3 vaginal examinations after ROM (46.3%), meconium-stained liquor (48.1%), and prolonged labour (36.1%). Maternal fever, foul-smelling liquor, and untreated or partially treated urinary tract infection were observed in 11.1%, 7.4%, and 7.4% of cases, respectively. Clinical signs suggestive of infection developed in 19 neonates, with 16 exhibiting more than one symptom within 72 hours. Respiratory distress (20.4%) was the most frequent presentation, followed by lethargy (15.7%) and poor feeding (13.0%). Other features included chest retractions (6.5%), delayed capillary refill (4.6%), tachycardia (4.6%), vomiting (2.8%), hypothermia (1.9%), and grunting (1.9%) (Table 2). Cord blood CRP ≥0.6 mg/dL was detected in 10 neonates (9.3%). At 24–36 hours of life, elevated neonatal CRP (≥0.6 mg/dL) was observed in 46 cases (42.6%), while CRP>1.2 mg/dL was noted in 22 cases. Blood culture positivity was confirmed in 4 neonates (3.7%) (Table 3). Elevated cord blood CRP showed a significant positive association with ROM>24 hours, prolonged labour, maternal fever,>3 vaginal examinations after ROM, foul-smelling liquor, and LSCS. At 24–36 hours, neonatal CRP ≥0.6 mg/dL was positively associated with primiparity, prolonged labour, maternal fever, foul-smelling liquor, meconium-stained liquor, and LSCS. When a higher CRP cut-off (>1.2 mg/dL) was applied, significant associations were observed with prolonged labour, maternal fever, meconium-stained liquor, and LSCS (Table 4). Based on clinical and laboratory evaluation, 4 neonates were classified as definite sepsis, 8 as probable sepsis, and 96 as no sepsis. Among neonates with cord blood CRP<0.6 mg/dL, subsequent CRP elevation>1.2 mg/dL at 24–36 hours was significantly associated with sepsis or probable sepsis (P<0.001). Neonatal CRP levels at 24–36 hours showed a statistically significant association with EOS (Table 5). Using a CRP cut-off of 0.6 mg/dL at 24 hours, the sensitivity, specificity, positive predictive value, and negative predictive value for diagnosing EOS were 83%, 64%, 23%, and 96.8%, respectively. Table 1 : Distribution of cases according to Sex, Weight, Gestational age, Parity of mother, Duration of rupture of membranes, Intrapartum risk factors, and Type of delivery Sex No. of Cases Percentage Male Female 64 44 59.3 40.7 Weight (kg) >2.5 <2.5 88 20 81.5 18.5 Gestational age (weeks) ≤37 15 13.9 >37 93 86.1 Parity of mother PRIMI 72 66.7 MULTI 36 33.3 Duration of rupture of membranes <12 hrs 30 27.8 12-24 hrs 58 53.7 >24 hrs 20 18.5 Intrapartum risk factors 1) MSL 52 48.1 2) >3 VE after ROM 50 46.3 3) Prolonged labour 39 36.1 4) Maternal fever 12 11.1 5) FSL 8 7.4 6) UTI 8 7.4 Type of delivery No.of Cases Percentage Normal 63 58.3 LSCS 45 41.7 Table 2. Shows the distribution of cases according to clinical features Clinical features No. of Cases Percentage 1. Respiratory distress 22 20.4 2. Lethargy 17 15.7 3. Poor feeding 14 13.0 4. Chest retraction 7 6.5 5. CFT>3 sec 5 4.6 6. Tachycardia 5 4.6 7. Vomiting 3 2.8 8. Hypothermia 2 1.9 9. Grunting 2 1.9 Table 3: Shows the distribution of cases according to the investigations done on the cord blood and investigations done on the neonatal blood between 24-36 hours No. of cases Percentage cord blood: Investigations CRP ≥ 0.6 mg/dl 10 9.3 Investigations: neonatal blood between 24-36 hours TLC<5000/cu.mm 8 7.4 ANC<1500/cu.mm 4 3.7 BCC>20% 5 4.6 CRP≥0.6mg/dl 46 42.6 Blood culture positive 4 3.7 Table 5 : Association between CRP levels and neonatal sepsis CRP Levels (mg/dl) Sepsis Probable Sepsis No sepsis P value Cord blood < 0.6 (n=98) At 24-36 hrs < 0.6 0 1(1.02%) 58(59.18%) 0.0205* ≥ 0.6 0 0 21(21.42%) 0.7943 > 1.2 3(3.06%) 3(3.06%) 12(12.24%) 0.0001** Cord blood >6 (n=10) At 24-36 hrs < 0.6 0 1(10%) 2(20%) 0.9999 ≥ 0.6 0 1(10%) 2(20%) 0.999 >1.2 1(10%) 2(20%) 1(10%) 0.3333 Table 4: Shows the distribution of cases according to the association of risk factors with CRP levels Risk factors Number of cases observed CRP (mg/dl) Risk ratio Cord Blood (≥0.6 mg/dl) (n=10) Neonatal Blood (≥0.6 mg/dl) (n=46) Neonatal Blood (>1.2 mg/dl) (n=22) Cord Blood (≥0.6 mg/dl) Neonatal Blood (≥0.6 mg/dl) Neonatal Blood (>1.2mg/dl) Primiparity 72 8 36 15 1.26 1.35 1.03 (80.0%) (78.3%) (68.2%) (0.91-12.63) (1.08-1.67) (0.78-1.35) ROM < 12 hrs 30 1 12 7 0.34 0.89 1.18 (10.0%) (26.1%) (31.8%) (0.07-1.64) (0.53-1.52) (0.66-2.15) ROM12-24 hrs 58 2 26 10 0.35 1.09 0.89 (20.0%) (56.5%) (45.5%) (0.12-1.00) (0.82-1.47) (0.54-1.23) ROM >24 hrs 20 7 8 5 5.27 0.89 1.30 (70.0%) (17.4%) (22.7%) (3.06-7.09) (0.46-1.77) (0.61-2.77) Prolonged labour 39 9 19 12 2.96 1.48 1.74 (90.0%) (41.3%) (54.5%) (2.17-3.98) (0.76-2.87) (1.15-2.63) Maternal fever 12 6 6 5 9.80 1.35 2.79 (60.0%) (13.0%) (22.7%) (4.5-21.3) (0.55-3.29) (1.16-6.73) >3VE after ROM 50 7 26 13 1.59 1.17 1.38 (70.0%) (56.5%) (59.1%) (1.1-2.35) (0.86-1.58) (0.96-1.96) FSL 8 2 6 3 3.27 4.04 2.35 (20.0%) (13.0%) (13.6%) (0.96-11.1) (1.09-14.91) (0.75-7.29) MSL 52 3 30 14 0.60 1.83 1.44 (30.0%) (65.2%) (63.6%) (0.27-1.35) (1.32-2.56) (1.03-2.01) UTI 8 1 2 1 1.40 0.45 0.55 (10.0%) (4.3%) (4.5%) (0.26-7.45) (0.12-1.66) (0.10-3.09) Male infant 64 5 25 12 0.83 0.86 0.90 (50.0%) (54.3%) (54.5%) (0.48-1.42) (0.66-1.14) (0.64-1.28) Normal delivery 63 3 24 10 0.49 0.83 0.73 (30.0%) (52.2%) (45.5%) (0.22-1.09) (0.63-1.09) (0.49-1.11) LSCS 45 7 22 12 1.80 1.28 1.42 (70.0%) (47.8%) (54.5%) (1.12-2.90) (0.83-2.01) (0.89-2.27) Risk ratio - > 1.2 – Positive association; 0.9-1.2 - Borderline positive association; < 0.9 - No association Table 5 : Association between CRP levels and neonatal sepsis CRP Levels (mg/dl) Sepsis Probable Sepsis No sepsis P value Cord blood < 0.6 (n=98) At 24-36 hrs < 0.6 0 1(1.02%) 58(59.18%) 0.0205* ≥ 0.6 0 0 21(21.42%) 0.7943 > 1.2 3(3.06%) 3(3.06%) 12(12.24%) 0.0001** Cord blood >6 (n=10) At 24-36 hrs < 0.6 0 1(10%) 2(20%) 0.9999 ≥ 0.6 0 1(10%) 2(20%) 0.999 >1.2 1(10%) 2(20%) 1(10%) 0.3333

Neonatal sepsis with its high mortality rate, still remains a diagnostic and treatment challenge for neonatal health care providers, developing countries have the highest incidence and mortality rates. This may be due to problems in utero by anatomical defects, infections and infected birth passage etc. Early diagnosis of neonatal septicemia helps the clinician in instituting antibiotics therapy at the earliest thereby reducing mortality in neonates. Early identification of an infected neonate also helps in abandoning unnecessary treatment of a non-infected neonate. In the present study an attempt has been made to document the effects of intrapartum risk factors for early onset sepsis on CRP levels in neonates and to assess the suitability of this test in the diagnosing EOS. Male babies were more than the female babies in the present study, showing a ratio 1.5:1. These results are comparable with observation by other authors. Male preponderance in the neonatal septicemias may be linked to the X-linked immonoregulatory gene resulting in the host’s susceptibility to the infection in males. In the present study the higher proportion of cases were with birth weight >2.5 kg. The results of other studies showed higher proportion of cases with birth weight <2.5 kg. As during our study there were less babies with weight<2.5kg delivered in the hospital, less cases were observed. In the present study the higher proportion of cases were found to with gestational age>37 weeks. The result of our study was comparable to the studies done by the Saxena.S et al5 etc The higher proportion of cases among the term neonates compared to the preterm neonates in our study probably reflect difference in the population characteristics and the occurrence of the predisposing factors(preterm incidence) among them. In the present study higher proportions of the mothers were primi gravida. The results of our study were comparable to the study done by Mathai et al. In the present study, duration of rupture of membranes<12hrs as a risk factors is 27.8% 0f cases which is comparable to study done by Mathai et al who showed 23.2% Duration of rupture of membranes was 12-24 hrs seen in which was comparable to study done by Mathai et al who showed 46.8%. Duration of rupture of membranes >24 hrs was seen in 18.5% as a risk factor which was comparable to studies done by Raghavan.et al6 and Mathai et al. FSL as a risk factors was observed in 7.4% cases, which was comparable to studies done by Dawodu et al7, Raghavan et al. MSL as a risk factor was observed in 48.1% cases, which was comparable to study done by Raghavan et al. Prolonged labour as a risk factor was observed in 6.1%% cases, which was comparable to studies done by Mathai et al,9 and Yancey et al. UTI as a risk factor was observed in 7.4%% cases, which was comparable to studies done by Betty Chacko et al9. The variations in the occurrence of intrapartum risk factors probably reflect differences in the rates of occurrence of the predisposing risk factors in the various studies. In the present study higher proportion of babies had respiratory distress, lethargy, poor feeding as predominant clinical features. The results of our study was comparable to the studies done by Dowadu et al and Jaswal R S et al.10 In the present study cord CRP was elevated (≥0.6 mg/dl) in 10 babies (9.3%). In the present study TLC<5000/ cu.mm is considered to be leucopenia. Akenzua et al11 reported that total WBC count were not helpful in diagnosing bacterial infection during first days of life. In the present study CRP ≥0.6mg/dl done at 24 hrs after birth in babies with neonatal risk factors was 42.6% which was comparable with the Mathai et al study. Absolute neutrophil count<1500/cu mm was observed in only 3.7% babies which was comparable with the Yadav A K et al study. Akenzua et al115 reported that absolute number of neutrophils were not helpful in diagnosing bacterial infection during first 5 days of life. The success of isolating bacterial pathogens from blood depends upon the quantum of blood cultured, frequency of culture and duration of incubation and the volume of blood required for the isolation of the pathogen depends on the magnitude of septicemia, which is directly related to the age of the patient. All the above reasons say why blood culture positivity is low. Dutta et al in their study showed that confirmation of the diagnosis by definitive culture is not rapid or particularly sensitive. Of all the new borns only 3-5 per 1000 admitted to a newborn intensive care unit have culture proven septicemia Referring to above table, The present study showed a positive association between elevated cord blood ≥0.6mg/dl and maternal risk factors like primiparity, ROM >24 hrs, prolonged labour, maternal fever,>3 VE after ROM, FSL, UTI and babies extracted by LSCS. This is comparable to the study done by Mathai et al which showed positive association between elevated CRP level with ROM >24hrs, prolonged labour. In present study, at 24 hrs elevation in CRP level ≥ 0.6mg/dl showed positive association with maternal risk factors like primiparity, prolonged labour, maternal fever, FSL, MSL and in babies extracted by LSCS. This is comparable to the study done by Mathai et al, which showed elevated CRP level ≥0.6mg/dl with primiparity,>3 VE after ROM. In the present study, at 24 hrs when the cut off CRP level was increased to >1.2mg/dl, showed positive association with maternal risk factors like ROM <12 hrs and > 24 hrs, prolonged labour, maternal fever,>3 VE after ROM, FSL, MSL and LSCS. Mathai et al showed in their study that when cut off of CRP was increased to>1.2mg/dl, positive association was noted only with maternal fever. Stimuli after the infection like hypoxia, trauma and metabolic changes can induce production of proinflammatory mediators. Significant association is reported between birth asphyxia and associated IL-6levels. In prolonged labour IL-6 level rise in the neonate probably related to physical activity of labour. This cytokine stimulates CRP production. In the present study, 21.42% cases with no evidence of sepsis had CRP levels >0.6mg/dl at 24 hrs which is comparable with Mathai et al study. 12.24% cases with no evidence of sepsis had CRP level≥1.2mg/dl at 24hrs which is comparable with Mathai et al study. Only 3.06% cases with sepsis with probable sepsis had CRP level>1.2mg/dl at 24 hrs which is comparable with Mathai et al study. From the above inference it can be stated that CRP elevated has no significant association with presence or number of signs. In the present study, single CRP value has sensitivity of 83%, specificity of 64%, postive predictive value 23% and negative predictive value of 96.7% which is comparable to observation made by Mathai et al and other studies. The difference in various studies is due to the different cut off value used in the qualitative test. We had higher sensitivity because our cut off was 0.6mg/dl.

Early and accurate diagnosis of neonatal sepsis is essential to reduce morbidity and mortality while minimizing unnecessary antibiotic exposure. Although blood culture remains the definitive diagnostic method, its delayed results and low sensitivity limit its utility in early clinical decision-making. CRP estimation is a rapid, cost-effective, and easily performed test that provides useful supportive information in the evaluation of EOS. This study demonstrates that intrapartum risk factors can independently elevate cord and neonatal CRP levels in the absence of infection. A CRP value<0.6 mg/dL at 24 hours has a high negative predictive value and may be useful in excluding EOS. While CRP should not replace clinical judgment or blood culture, its cautious use as part of a combined clinical and laboratory approach may help reduce unnecessary antimicrobial therapy and improve neonatal outcomes.

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