Abbreviations

Antigen-presenting cells: (APCs), bronchopulmonary dysplasia: (BPD), C-reactive protein: (CRP), early-onset neonatal sepsis: (EONS),  gestational age: (GA), lipopolysaccharide: (LPS), late-onset neonatal sepsis: (LONS), Preterm infants: (PTs) Radioimmunoassay: (RIA), T helper: (Th), Tumor necrosis factor-alpha: (TNF-α), interleukin: (IL), necrotizing enterocolitis:  (NEC), neonatal intensive care unit: (NICU), Parathyroid hormone: (PTH), vitamin D receptors: (VDRs).

Acknowledgements:

We would like to thank all medical staff at NICU of Minia university hospital for their support.

Competing interests

The authors declare that no conflict of interest Funding

No funding agency is providing us with assistance for this project in the form of funds.

Authors' contributions

NE and AG: Study design and analysis, interpretation of data, drafting of the manuscript and revision of the manuscript for important intellectual content. AM: laboratory data interpretations and Statistical analysis. All the authors reviewed and approved the final manuscript; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Date received: 17^th May 2024, accepted 30^th June 2024

Introduction

Neonatal sepsis is a clinical syndrome of systemic illness accompanied by bacteremia occurring in the first month of life. [1-3] Neonatal sepsis is a medical entity affecting infants under 28 days of age with signs of systemic infection and isolated infections in the bloodstream.[4] Sepsis in neonates have an incidence of 1-7/1000 live births and 12-27/1000 live births for infants < 1500g and mortality is 13-25%.[4] and increased rates in premature neonates classified as severe sepsis when it is associated with organ dysfunction.[5] Sepsis is divided into two types ;Early onset sepsis(EONS) which develops in the 1st week of life but it is sometimes restricted to the first three days  of postnatal life ,usually multisystem disease with severe respiratory symptoms and high mortality rate.[6] Early onset sepsis acquired from  genital tract intrapartum and usually associated with chorioamnionitis.[7]  while  late  onset sepsis occur as early as five days but it is most common after the first week of life and less association with obstetric complications,[8,9] and usually have a known cause , often meningitis or sepsis and its usually acquired from    the environment, Staphylococci account for 30 to 60% of LONS cases and are  due to intravascular devices (particularly central vascular catheters). [11,12]

E.coli is       also a significant cause of late-onset sepsis, especially in extremely preterm infants.[13,14] Neonatal sepsis is diagnosed by poor suckling, skin mottling and respiratory distress, laboratory markers as elevated C- reactive protein(CRP) , positive blood cultures for E. Coli and Staphylococci or other organisms. [23-25], also contaminated ryle tubes may be a cause for introduction of organinsms. [26] While contaminated respiratory equipment is suspected in outbreaks of hospital- acquired Pseudomonas pneumonia among preterms. [27] Although  screening and intrapartum antibiotic prophylaxis for group B streptococcus have significantly lowered the rate of early-onset sepsis due to this organism, the rate of late-onset GBS sepsis has remained unchanged, which is consistent with the hypothesis that LONS is usually acquired from the environment. [28] The role          of anaerobes as Bacteroides fragilis in LONS in preterm infants remains unclear, although deaths among   preterms   were   due to Bacteroides bacteremia. [29] Also candida species are important causes of LONS, occurring in 10 to 18% of preterms. [30] Vitamin D is an essential for neonate's especially preterm ones it is important for calcium &phosphate metabolism, for immunity, endothelial function, and antimicrobial activity [14]. Immune system cells express vitamin D receptors (VDRs), vitamin D is essential for immune system regulation [15]. It regulates the balance between inflammation and tissue damage by promoting lymphocyte differentiation. [16] Vitamin D decrease the production of cytokines as interleukin (IL)-2, IL-6, IL-8, and tumor necrosis factor-α (TNF-α). [17] Also the immune cells including monocytes and antigen-presenting cells (APCs), express vitamin D receptors. [18]

Vitamin D promotes the transition in the immune system from helper T (Th1) and Th17 to Th2 and regulatory T cells. [19] Vitamin D also improves intestinal health and homeostasis by decreasing intestinal damage by lipopolysaccharide (LPS). [20] Vitamin D deficiency in preterm infants can weaken the immune system, alter hormonal metabolism, and cause the spread of various diseases, infections, and serious illnesses all of which can be fatal.

Patients and Methods

This Prospective Cohort study conducted at NICU(neonatal intensive care unit) in Minia University Hospitals onto Sixty five preterms≤30-32 weeks’  GA and/or≤1500 g birth weight (Group I) with inclusion criteria and other fifty term healthy babies represent( Group II )with measured vitamin D serum levels at birth. Babies were excluded if they did not matches the age and weight at birth established, also if mortality before 36 weeks, Chromosomel abnormalities, Genetic anomalies or Congenital malformations. We carried out a follow-up on infants at one month and grouped into two subgroups for tests of association with clinical outcomes based on vitamin D blood levels: Group Ia with low vitamin D Baseline < 20 ng/ml , Group Ib with vitamin D Baseline > 20 ng/ml, according to the criteria of the Endocrine Society Clinical Practice Guidelines.[18]

Collection of data and definitions:

Demographic data, including data on pregnancy and delivery were collected from the mothers. All preterm neonates newly diagnosed with LONS between Feburary 2024 and May 2024 participated in this study. Diseases of prematurity were noted: sepsis, hyaline membrane disease, bronchopulmonary dysplasia (BPD), intraventricular hemorrhage, periventricular leukomalacia, retinopathy of prematurity and necrotizing enterocolitis (NEC). [35] Late onset sepsis (LOS) is defined as occurance of signs and symptoms of severe sepsis after three days of life. [42-45].  

Methodology:

Blood sampling protocol:  venous blood samples obtained from neonates under complete aseptic condition at 28 days of life and at one month for  the following routine and specific investigation ,include Blood culture done using (BD™ BACTEC™ FX40 Automated Blood Culture System, Becton Dickinson, USA) and subcultures done for positive cases to identify the causative organism [identification and AST done by (VITEK-2, bioMérieux -France)]. Complete blood count, with differential determined by automated cell counter, Sysmex KX-21N (TAO Medical incorporation, Japan). Separated sera were used for  measuring CRP by kinetic method (by GENRUI,Biotec Inc.in china.),according to the manufactures instruction.The remaining serum was preserved at -80°c until determination of  serum level of vitamin D and parathyroid hormone(PTH) by fully automated Chemiluminescence technology (cobas E 411-Roche Diagnostic GmbH Germany). Serum calcium and phosphorus were assessed by (Mindray-BS-800M, China).

Ethical consent

Ethics approval: The ethics committee for the faculty of medicine authorized this research. Our study was carried out in accordance with the Declaration of Helsinki and approved by the Institutional Hospital Ethical Committee number 1059-01-2024. Participants Informed written consent was obtained from their parents or legal guardians. Maintain the participant's privacy and anonymity, avoiding the use of misleading tactics, allowing participants to leave our study at any time, a release for publishing was obtained.

Availability of data and materials: The corresponding author may be reached for a reasonable request for the datasets utilized and/or analyzed in the present work.

Statistical Analysis:

Data         were    collected,       tabulated and statistically analyzed using                an IBM compatible personal computer with Statistical Package for the Social Sciences (SPSS) version 26 (SPSS Inc. Released 2018. IBM SPSS statistics for windows, version 26.0, Armnok, NY: IBM Corp.) non-numerical         data        were given as percentages, whereas numerical variables were expressed as means and standard deviations. Using two-tailed t-tests, the variations between both groups      were calculated. P-values below 0.05 were regarded as significant. The correlation coefficient of Pearson determined the magnitude     of correlations.    Using                                  the statistical software Prism 3.0, all data were evaluated (Graph Pad Software, USA: San Diego, CA). Microsoft Office Excel 2016 was utilized to calculate the numbers.

Results

Regarding demographic data of groups, there were no differences observed between the control group and the PTs group in relation to the age of the mothers and sex of infants. Table 1

Regarding biochemical data of groups there were no differences in vitamin D serum levels at birth when comparing PTs to term infants, or regarding maternal levels. Table 2

There was a positive correlation regarding vitamin D serum levels of mother/ infant (r: 0.466; p < 0.001) in both groups. Regarding the biochemical data analyzed; increased levels of PTH were observed in the PTs group. We found differences in the levels of calcium and phosphorus among groups; such did not have clinical relevance as values were still within the normal range. Table 2

A comparative analysis was conducted to identify factors associated with vitamin levels at birth and we found that vitamin D levels were dependent on the mother’s levels. Considering a cut-off point of 20 ng/ml for 25(OH) D, PTs (%) had vitamin D serum levels < 20 ng/ml and PTs (%) had vitamin D levels > 20 ng/ml. Table 3

The most frequent co-morbidities in preterm babes were LOS (48%), when the two subgroups of PTs were arranged according to Vitamin D and PTH levels (vitaminD<20ng/ml and PTH>60pg/ml versus vitamin D>20ng/ml and PTH<60pg/ml). Table 2

At one month of age; Group Ia with low was 49 babies while 16 infants were in group Ib with vitamin D Baseline > 20 ng/ml, the incidence of LOS was higher in the subgroup with low vitamin D levels <20 and PTH>60 Table 3

We demonstrated that vitamin D levels at one month of age were independently associated with LOS (P <0.025). Table 3 In preterm groups with low vitamin D show sepsis markers as positive CRP more than 6 mg/dl, high total white blood cell count, positive blood cuture (for staphaloccoci, E.Coli &for other organisms) Table 4

No correlation was observed between levels of PTH (r: 0.065; p: 0.545), calcium (r: 0.024; p: 0.816), or phosphorus (r: - 0.039; p: 0.721) with vitamin D. Table 5

Correlation was observed between incidence of LONS (r: - 0.16 p: 0.001), length of hospital stay (r: 0-.77; p: 0.001) with vitamin D. Table 5.

The receiver-operating characteristic (ROC) curve for serum vitamin D as a predictor of LONS, it shows an area under the curve (AUC) of 0.82 with sensitivity of 78.1% and specificity of 61.4% at cut off value for serum vitamin D of < 20.5 ng/ml (Figure 1).

Discussion

Late onset neonatal sepsis in preterm infants was associated with low vitamin D levels, below 20 ng/mL in cord blood because these levels are associated by abnormal monocyte responses that make newborns more vulnerable to infections. [42-45]

Regarding demographic data of our study groups    there  were no differences observed between the full term infants and the PTs group in relation to the age of the mothers and sex of infants also for biochemical data of groups there were no differences in vitamin D levels at birth when comparing PTs to term infants, or regarding maternal levels. We found a positive correlation for vitamin D serum levels of mother/ infant (r: 0.466; p < 0.001) in both groups and we found that vitamin D levels were dependent on the mother’s levels.

Cariolou M, et al.(2019)studies have described that maternal vitamin D serum levels < 20 ng/mL is a risk factor for neonatal poor outcomes,also suggested that pregnant females  should have vitamin D serum levels > 35 ng/mL for good  maternal and neonatal outcomes.[43]

Our results showed that in PTs, higher PTH levels at birth and these did not reach a normal range until one month of age, also this was in agreement with study of Cariolou M ,et al.,(2019) that indicate the vitamin D levels only does not define its deficiency or normal state  and that the vitamin D-PTH relationship must be cosidered  as PTH is a major hormone of bone resorption, and its serum levels are indicator for secondary hyperparathyroidism and metabolic bone disorders in extremely low birth weight neonates and preterms.[44,45]

We found  differences in  the levels of calcium and phosphorus among the term and preterm; such did not have clinical relevance as values were still within the normal range also there was no correlation between calcium (r: 0.024; p: 0.816) or phosphorus (r: -0.039; p: 0.721) levels and vitamin D. LOS incidence varies between 20% to 38% with PT in the first 120 days of life[45] while the high prevalence of LOS in our study population (48%) may be due to the choice of PT who is more risk for infections due immature immune system. [45]

We carried out a follow-up on infants at one month and classed them into two subgroups for tests of association with clinical outcomes based on vitamin D blood levels; Group Ia with low vitamin D Baseline < 20 ng/ml and there were forty nine babies while Group Ib with vitamin D baseline > 20 ng/ml were sixteen ones Analysis of our results after 1 month showed that decreaed vitamin D and high PTH levels were associated with more  LOS levels in PT and this in agree with the research of Mao Zedong, etal., (2018) reported more vitamin D deficiency in critical illness in preterm infants such as hyaline membrane disease, NEC, patent ductus arteriosus, and sepsis which are common in PT in the first days of life. Therefore, PTs admitted to the NICU are at higher risk for vitamin D deficiency. [45]

Laboratory markers for sepsis as high CRP ,leucocytosis and postive blood cultures were reported  and incidence of LOS after 1 month of observation was higher in the group with low vitamin D level and we showed that vitamin D at one month of age was independently associated with LOS (P< 0.025) and did not affect the rate of breastfeeding and the different exposures to ventilators while In study reported by Çetinkaya M et al. (2015), it was found that the lower the blood concentration of vitamin D, the higher the risk of EOS. [46] also Çetinkaya M et al. (2015) reported that pregnant women and their babies should reach higher vitamin D levels to prevent poor outcomes, and that vitamin D deficiency increases the risk of abortions and EOS and recommended measuring levels during pregnancy. [46] While, in another study, Hollis BW (2005) discovered that preterm with EONS had significantly lower vitamin D concentrations as well as discovered a positive correlation between vitamin D and high CRP, TNF-α, and IL-6 [38]. longer hospital stays in our study for PTs who developed sepsis was associated with delayed advances of enteral feeding in this group and also correlation was observed between incidence of LONS(r: - 0.16 p: 0.0001), length of hospital stay (r: 0-.77; p:0.001) with vitamin D levels in neonates especially preterm ones, and this is consistent with the study of Gäddnäs FP et al. (2020) determined total central vein catheter exposure and parenteral nutrition days as risk factors for sepsis and stated that breastfeeding is protective. [46]

In contrast to our results, study of WorknehBitew Z, etal., (2020) sepsis markers such as positive CRP, total white blood count and positive blood count were higher in the l full term infants with critical illness (p < 0.05) and reported decreased vitamin D levels in their mothers and stated that there is a positive relationship between vitamin D concentrations in infants and parents. [35] Our results showed that serum vitamin D as a predictor of LONS has a sensitivity of 78.1% and specificity of 61.4% at cut off value for serum vitamin D of < 20.5 ng/ml,While Jones T, et al.(2010)found that the ROC curve of specificity and sensitivity of vitamin D in the prediction of early onset sepsis (EONS), the sensitivity was 100%, the specificity was 73%, the positive predictive value was 73 at a vitamin D cut-off value of 20 ng/mL.[47,48]

Results have been reported by Tayel S, et al. (2018) in this point,It is important to establish the optimal dose of vitamin D intake among hospitalized PTs to achieve adequate vitamin D levels and prevent adverse outcomes. [8]

Limitation: limitations   of   the present study include small sample size

Conclusions

Preterm infants who have vitamin D deficiency with levels < 20 ng/ml have an increased chance of developing LONS and more severe outcomes.