European Journal of Cardiovascular Medicine

Neonatal sepsis is a life-threatening condition characterized by systemic infection and an exaggerated inflammatory response in newborns. It is a leading cause of morbidity and mortality, particularly in preterm and low-birth-weight infants [1]. Despite advancements in neonatal intensive care, the incidence of neonatal sepsis remains high, especially in developing countries, where healthcare-associated infections pose an additional burden [2]. Pathogens such as Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus are frequently implicated in neonatal sepsis [3]. Early-onset neonatal sepsis (EONS) occurs within the first 72 hours of life and is primarily acquired through vertical transmission from the mother, whereas late-onset neonatal sepsis (LONS) is often hospital-acquired and manifests after 72 hours [4].

The gut microbiota plays a crucial role in neonatal immune system development and pathogen resistance. Alterations in the gut microbiome composition have been linked to an increased risk of neonatal infections and inflammatory diseases [5]. Probiotics, which are beneficial live microorganisms, have been proposed as a potential intervention to support gut health, enhance immune

function, and prevent neonatal sepsis [6]. Various clinical

trials have explored the efficacy of probiotics in reducing the incidence of sepsis, with promising results [7]. Probiotics exert their beneficial effects through multiple mechanisms, including competitive inhibition of pathogenic bacteria, enhancement of intestinal barrier integrity, and modulation of the host immune response [6,7].

Despite promising data, the use of probiotics in neonatal care remains a topic of debate due to variability in probiotic strains, dosages, and treatment durations across different studies. Furthermore, safety concerns, including potential probiotic translocation and sepsis, necessitate rigorous evaluation before widespread clinical adoption. Given these considerations, this study aims to investigate the role of probiotics in preventing neonatal sepsis in a tertiary care setting over a six-month period.

Aims and Objectives

Aim

The primary aim of this study is to evaluate the efficacy of probiotic supplementation in preventing neonatal sepsis among preterm and low-birth-weight neonates admitted to a tertiary care center.

Objectives

1. To determine the incidence of neonatal sepsis in probiotic-supplemented neonates compared to a control group.
2. To evaluate the impact of probiotic supplementation on inflammatory markers such as C-reactive protein (CRP) and Procalcitonin.
3. To investigate the mortality and morbidity rates, including the incidence of necrotizing enterocolitis (NEC) and other complications.

Study Design and Setting

This prospective, randomized controlled trial was conducted over six months at a tertiary care centre.

Sample Size Calculation

Using the standard formula for comparative studies, a total of 500 neonates were recruited, with 250 assigned to the probiotic group and 250 to the control group.

Inclusion Criteria

·        Preterm neonates (<37 weeks gestation)

·        Low birth weight (<2500 g)

·        No major congenital anomalies

·        No prior antibiotic administration before enrollment

Exclusion Criteria

·        Severe congenital malformations

·        Neonates requiring immediate intensive care

·        Parental refusal to participate

Intervention

Neonates in the probiotic group received Lactobacillus rhamnosus GG (LGG) and Bifidobacterium breve orally once daily for the first 28 days of life. The control group received standard care without probiotics.

Outcome Measures

Primary outcome: Incidence of neonatal sepsis diagnosed based on clinical signs, blood culture, and inflammatory markers (CRP >10 mg/L, Procalcitonin >2 ng/mL).

Secondary outcomes: Duration of hospital stay, mortality rate, and adverse effects of probiotics.

Statistical Analysis

Data were analyzed using SPSS software. Chi-square tests were used to compare categorical variables, and a p-value <0.05 was considered statistically significant.

1. Study Population and Baseline Characteristics

A total of 500 neonates were enrolled in the study, with 250 assigned to the probiotic group and 250 to the control group. Both groups were comparable in terms of key demographic and clinical characteristics, ensuring a balanced distribution of potential confounding factors.

The mean gestational age was similar between the two groups, with a slight variation that was not statistically significant (p = 0.72). Similarly, the mean birth weight did not differ significantly between the probiotic (1780 ± 320 g) and control (1765 ± 310 g) groups (p = 0.65).

The sex distribution was nearly identical, with 55% males in the probiotic group and 54% males in the control group (p = 0.81). A higher proportion of neonates were delivered via cesarean section (C-section) in both groups, but the difference was not statistically significant (p = 0.74).

Regarding maternal factors, antenatal steroid use was observed in 72% of mothers in the probiotic group and 71% in the control group (p = 0.83), while maternal infections were present in 12% and 14% of cases, respectively (p = 0.62). These results indicate that both groups were well-matched at baseline.

A summary of these findings is presented in Table – Baseline Characteristics of Study Groups below.

Table1 – Baseline Characteristics of Study Groups

2. Incidence of Neonatal Sepsis and Microbiological Profile

The incidence of neonatal sepsis was significantly lower in the probiotic group (10%, 25/250) compared to the control group (22%, 55/250) (p < 0.001). The relative risk reduction (RRR) for sepsis with probiotic use was 54.5%, demonstrating a strong protective effect. The Chi-square test confirmed a statistically significant difference (χ² = 12.34, p < 0.001).

Among sepsis cases, early-onset neonatal sepsis (EONS) accounted for 40% of infections, whereas late-onset neonatal sepsis (LONS) comprised 60%. A higher proportion of LONS was observed in the control group, suggesting a possible protective role of probiotics in hospital-acquired infections.

The microbiological analysis of culture-positive cases identified the following common pathogens:

Table 2:Pathogen distribution among sepsis cases

The rate of multidrug-resistant (MDR) infections was lower in the probiotic group (12% vs. 16%), but the difference was not statistically significant (p = 0.48). These findings suggest that probiotics may contribute to reducing overall sepsis rates, particularly in preventing late-onset infections.

Figure1: Incidence of Neonatal Sepsis in Both Groups

Figure 1.Incidence of neonatal sepsis in both groups. The probiotic group showed a significantly lower incidence of neonatal sepsis (10%) compared to the control group (22%) (p < 0.001). This supports the potential role of probiotics in reducing the risk of neonatal sepsis.

3. Inflammatory and Haematological Markers

Inflammatory markers were assessed in septic and non-septic neonates. C-reactive protein (CRP) and Procalcitonin levels were significantly higher in neonates with sepsis, with lower levels observed in the probiotic group.

Table3: Comparison of Inflammatory and Haematological Markers between Study Groups

The probiotic group demonstrated significantly lower inflammatory marker levels, suggesting a protective effect in reducing the systemic inflammatory response associated with neonatal sepsis. This reduction in inflammatory markers was accompanied by improved clinical outcomes, including a lower incidence of sepsis-related complications and a shorter duration of hospital stay in the probiotic group compared to the control group.

Statistical Analysis: Mean difference with 95% confidence intervals (CRP: 6.8 mg/L [95% CI: 5.7–7.9], Procalcitonin: 1.4 ng/mL [95% CI: 1.1–1.7], WBC: 3.3 ×10³/µL [95% CI: 2.2–4.1], Platelet count: 35 ×10³/µL [95% CI: 28–42]) confirmed significant differences between groups.

Figure2: Box Plot of CRP and Procalcitonin Levels in Both Groups

Comparison of inflammatory marker levels between probiotic and control groups. The probiotic group exhibited significantly lower CRP and Procalcitonin levels, indicating a potential reduction in systemic inflammation associated with neonatal sepsis (p < 0.001). Error bars represent interquartile ranges.

4. Clinical Outcomes, Mortality, and Morbidity

Clinical outcomes, including hospital stay duration, intensive care requirements, mortality rates, and morbidity incidences, were assessed to evaluate the overall impact of probiotic supplementation in neonatal care. Significant differences were observed between the probiotic and control groups, particularly in hospital stay length and mortality reduction.

The mean hospital stay duration was significantly shorter in the probiotic group compared to the control group, suggesting improved recovery rates. Additionally, the need for intensive care unit (ICU) admissions and ventilator support was lower among neonates receiving probiotics, reflecting a potential protective effect against severe complications.

Mortality analysis revealed a statistically significant reduction in overall and sepsis-related deaths in the probiotic group. Furthermore, the incidence of necrotizing enterocolitis (NEC) and other major morbidities was notably lower, reinforcing the hypothesis that probiotic supplementation may enhance neonatal health outcomes. The statistical significance of these findings was confirmed using the Student’s t-test for hospital stay duration and relative risk reduction (RRR) analysis for mortality rates.

The following sections present detailed results and statistical comparisons, supported by graphical representations.

• Hospital Stay Duration: The probiotic group had a significantly shorter hospital stay (15.2 ± 3.1 days) compared to the control group (20.5 ± 4.2 days, p < 0.001).
• ICU Admissions and Ventilator Support Requirement: The need for ICU admission was 18% in the probiotic group vs. 32% in the control group (p = 0.002). Ventilator support was required in 10% of probiotic group neonates vs. 20% in the control group (p = 0.01).
• Mortality Rate: Overall mortality was 4% in the probiotic group compared to 9% in the control group (p = 0.03). Sepsis-related mortality was significantly lower in the probiotic group (2% vs. 6%, p = 0.02).
• Morbidity Rates: The incidence of necrotizing enterocolitis (NEC) was lower in the probiotic group (3%) compared to the control group (8%, p = 0.01).
• Statistical Analysis: Student’s t-test confirmed a significant reduction in hospital stay, while relative risk reduction for mortality was 55.6%.

Figure3: Line graph – Hospital stay duration in both groups

Figure 4:Mortality and Major Morbidities in Neonates

Distribution of mortality and major morbidities in neonates. The majority of neonates survived without necrotizing enterocolitis (NEC), while a smaller proportion developed NEC or experienced mortality. Sepsis-related mortality was notably lower in the probiotic group. Percentages represent the proportion of each outcome category within the study population.

5. Safety and Adverse Effects of Probiotic Use

The safety profile and tolerability of probiotic supplementation were assessed by monitoring adherence, tolerance, and any adverse effects observed during the study.

Tolerance and Adherence: Among neonates in the probiotic group, 95% (238/250) successfully completed the full probiotic course without any issues, while 5% (12/250) experienced mild feeding intolerance leading to early discontinuation.

Adverse Events: No cases of probiotic-associated infections or severe adverse effects were reported. Mild feeding intolerance was observed in 6% (15/250) of neonates in the probiotic group compared to 4% (10/250) in the control group (p = 0.32, not significant).

Table4 – Adverse effects in probiotic vs. control groups

These findings suggest that probiotic supplementation was well-tolerated in preterm neonates, with no significant increase in adverse events compared to the control group. The mild feeding intolerance observed was transient and did not lead to severe complications.

6. Summary of Key Statistical Findings

The findings of this study demonstrate a significant reduction in neonatal sepsis and mortality rates in the probiotic-supplemented group.

Incidence Reduction of Neonatal Sepsis: The probiotic group showed a 54.5% relative risk reduction in neonatal sepsis compared to the control group (p < 0.001). The 95% confidence interval (CI) for this reduction ranged from 42.1% to 66.9%, confirming a strong protective effect.

Mortality Risk Reduction: The overall mortality rate was significantly lower in the probiotic group (4% vs. 9% in control, p = 0.03), with a relative risk reduction of 55.6%. Sepsis-related mortality was also lower in the probiotic group (2% vs. 6%, p = 0.02).

These results suggest that probiotic supplementation in preterm neonates significantly reduces the risk of sepsis and mortality, supporting its potential role as a preventive strategy in neonatal care.

The results of this study provide compelling evidence that probiotic supplementation significantly reduces the incidence of neonatal sepsis. Our findings align with previous research demonstrating the protective effects of probiotics in preterm neonates. For instance, Nair &Soraisham (2013) [8] reported that probiotics effectively prevent nosocomial sepsis, which corroborates our observed 54.5% relative risk reduction in neonatal sepsis incidence (p < 0.001). Similarly, Dani et al. (2002) [9] noted a significant reduction in bacterial sepsis and urinary tract infections in preterm infants receiving probiotics, reinforcing our findings of reduced systemic infections in the probiotic group.

The reduction in inflammatory markers, such as CRP and Procalcitonin, in our study is consistent with prior findings. Kulkarni et al. (2022) [10] demonstrated that probiotic administration lowers systemic inflammatory markers, which aligns with our findings of significantly lower CRP (8.6 ± 3.2 vs. 15.4 ± 4.1 mg/L, p < 0.001) and Procalcitonin

levels (1.8 ± 0.7 vs. 3.2 ± 1.2 ng/mL, p < 0.001) in the

probiotic group compared to the control. The immunomodulatory effects of probiotics likely contribute to this anti-inflammatory response, as previously suggested by Hu et al. (2017) [11], who emphasized the role of probiotics in modulating cytokine production and gut immune responses.

A key outcome of our study was the shorter hospital stay duration in the probiotic group (15.2 ± 3.1 vs. 20.5 ± 4.2 days, p < 0.001). This reduction in hospitalization may have significant economic implications, potentially lowering overall healthcare costs by reducing the need for prolonged intensive care and associated medical interventions. Future studies should explore the cost-effectiveness of probiotic supplementation in neonatal care to further validate these potential benefits. This reduction mirrors findings from Baucells et al. (2016) [12], who reported that probiotic supplementation in preterm neonates was associated with decreased hospitalization due to lower infection rates. The reduced length of hospital stay in our study suggests improved overall neonatal health and faster recovery, highlighting the cost-effectiveness of probiotic use in neonatal care settings.

Our study also found a 55.6% reduction in overall mortality risk in probiotic-supplemented neonates, with sepsis-related mortality significantly lower (2% vs. 6%, p = 0.02) in the probiotic group. This aligns with findings from AlFaleh&Anabrees (2014) [13], who concluded that probiotics significantly decrease mortality in preterm infants by reducing sepsis severity. Similarly, Sinha et al. (2021) [14] in their randomized trial demonstrated a comparable reduction in sepsis-related mortality, further supporting our conclusions.

The safety profile of probiotics in our study was favourable, with no probiotic-associated infections or severe adverse effects reported. Mild feeding intolerance occurred in 6% of probiotic-supplemented neonates versus 4% in controls (p = 0.32), but this was not statistically significant. These findings align with Dani et al. (2016) [15], who reported no severe adverse effects associated with probiotic use. However, Cavicchiolo et al. (2020) [16] highlighted isolated cases of Lactobacillus-associated sepsis in extremely preterm infants, reinforcing the need for strain-specific safety assessments and proper clinical monitoring.

Despite these promising results, our study has some limitations. Being a single-centre trial, the findings may not be generalizable to all neonatal populations. Additionally, a longer follow-up period is needed to assess long-term outcomes of probiotic supplementation. Seghesio& De Geyter (2021) [17] emphasized the need for multicentre trials to establish standardized probiotic regimens, a recommendation that aligns with our call for future research to optimize probiotic use in neonatal care. Key factors requiring standardization include the selection of probiotic strains, optimal dosage, duration of administration, and the timing of initiation. These variables can significantly influence efficacy, and inconsistencies in previous studies highlight the need for well-defined protocols to maximize clinical benefits while minimizing risks.

Our study strongly supports the role of probiotics in reducing neonatal sepsis incidence, lowering inflammatory markers, shortening hospital stay, and improving survival outcomes. Given the statistical significance and clinical relevance of these findings, probiotic supplementation should be considered a preventive strategy in high-risk neonates. Further multicentre trials are essential to confirm these results and establish standardized protocols for probiotic administration in neonatal intensive care units.

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