Role of Vitamin D and Parathyroid Hormone Levels in Prediction of Late Onset Sepsis among Preterm Infants: A Prospective Cohort study

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. The authors have personally sponsored the research

Authors' contributions

NE and AG: Study concept and design and analysis, interpretation of data, drafting of the manuscript and critical 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 condition 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 rate is 13-25%.[4] and higher rates in premature babies , which was classified as severe sepsis when it combined with organ dysfunction.[5]

Sepsis is divided into two types ;Early onset sepsis(EONS)  occur which develops before 7 days of life but is sometimes restricted to the first 72 h of postnatal life ,usually multisystem disease  with severe respiratory symptoms and high mortality rate.[6]Early onset sepsis  acquired from maternal genital tract intrapartum and associated with chorioamnionitis.[7] while late onset sepsis occur as early as 5 days but is most common after the first week of life and  less association with obstetric complications,[8,9]and usually have a known focus, often meningitis or sepsis and its usually acquired from the environment, Staphylococci account for 30 to 60% of LONS cases and are most frequently due to intravascular devices (particularly central vascular catheters), [11,12]E. coli is also becoming increasingly recognized as 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 high C-reactive protein(CRP) ,positive blood cultures for E.Coli and Staphalcocci or other organisms .[23-25],also Isolation of many organisms from blood or cerebrospinal fluid may be due to contaminated feedings. [26] While contaminated respiratory equipment is suspected in outbreaks of hospital-acquired Pseudomonas aeruginosa pneumonia among preterm babies. [27]

Although universal screening and intrapartum antibiotic prophylaxis for group B streptococcus have significantly decreased the rate of early-onset disease 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 Bacteroidesfragilis 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 preterm infants. [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].

Vitamin D regulates the balance between inflammation and tissue damage by promoting lymphocyte differentiation. [16] Vitamin D decrease the production of pro-inflammatory cytokines as interleukin (IL)-2, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) and some reactions inflammatory cytokines, such as IL-10, are produced. [17] Also the immune system 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 intestinal homeostasis by reducing intestinal damage caused 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. [20-23] Also in critically ill preterm infants, vitamin D deficiency increases the risk of sepsis and death resulting from renal excretion of vitamin D metabolites, while co morbidity and nutritional supplementation also affect vitamin D status in acute disease. [32] 

Babies with vitamin D deficiency have a lower CD4/CD8 ratio, which is a sign of immunity. [16-20] Vitamin D prevents dendritic cell maturation and differentiation by interacting with the vitamin D receptor (VDR). [33.34]

Vitamin D inhibits B cell function by altering the response of CD4 T lymphocytes and inhibiting the release of monocyte/macrophage cytokines, thus enhancing immunity. [35, 36] In sepsis, vitamin D will affect coagulation, endothelial function and hemodynamic stability, as well as vascular permeability. [37-38] Neonatal Intensive Care Units (NICU) Sepsis mortality rate in preterm infants can reach 21.9%. On the other hand, there is vitamin D deficiency in most neonatal intensive care units (NICUs) around the world It is between 35-70% in premature babies [39,40]   who are more likely to suffer from vitamin D deficiency than term babies. [40] 

Despite improvements in sanitation, the development of new powerful antibiotics, and improved diagnostic techniques, sepsis remains a leading cause of infant death (accounting for 30–50% of all infant deaths in developing countries). [41, 42] 

Preterm neonates with symptoms of LONS should have a complete blood count, differential smear, blood culture, urine culture, and lumbar puncture (LP) if medically necessary should be done as soon as possible.[43]  The diagnosis is confirmed by isolating the organism in culture and finally babies should receive comprehensive treatment with antibiotics. [44]

Aim of the work: To examine the relationship between vitamin D and delayed onset sepsis in preterm babies.  Patients and Methods

We conducted a Prospective Cohort study, Sixty five preterms≤30-32 weeks’ GA and/or≤1500 g birth weight (Group I) with inclusion criteria  and other fifty term  healthy babies Group II) and measured vitamin  D serum levels at birth.Babies were excluded if they did not fulfils the age and weight at birth established, also if mortality before 36 weeks, Chromosome abnormalities, Genetic anomalies or Congenital malformations. We carried out a follow-up on infants at one  month and divided 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 , Group Ib with vitamin D Baseline > 20 ng/ml, following the criteria provided by the Endocrine Society Clinical Practice Guidelines.[18]

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.

Collection of data and definitions:

Demographic and clinical data, including data on pregnancy and delivery were collected from the mothers, term infants and PTs. All preterm neonates newly diagnosed with LONS between Feburary 2024 and May 2024 participated in this study. Diseases related to prematurity were noted: sepsis, hyaline membrane disease, bronchopulmonary dysplasia (BPD), intraventricular hemorrhage, periventricular leukomalacia, retinopathy, and necrotizing enterocolitis (NEC). [35]

Late onset sepsis (LOS) is defined as the development of signs and
symptoms of severe sepsis after 72 hours of life. [42-45]

 Setting:

The research was conducted in Minia City, Egypt (population total of 8 million as of December 31, 2023). Only Minia University Hospital in Minia City provides a neonatal intensive care unit and tertiary-level care. Therefore, the number of preterms managed at the university hospital in Minia can be used to determine the prevalence of LONS in this cohort.

Blood sampling: Blood samples from neonates were obtained from venous blood and drawn into BD Vacutainer® SST (BD)tubes. At 28 days of life and at one month, the blood samples of the PTs were obtained from a peripheral vein as part of the routine protocol followed in the unit to monitor metabolic status

Sepsis work up include blood culture, complete blood cell count with differential and C-reactive protein (CRP) at one month of age

Umbilical vessels are frequently contaminated by organisms on the umbilical stump, especially after a number of hours, so blood cultures from umbilical venous lines may not be reliable. Therefore, blood for culture should be obtained by venipuncture, preferably at 2 peripheral sites.

The blood collected into these tubes was allowed to clot in the cold
for 30 min prior to centrifugation at 1500 x g for 10 min at 4 °C. Once centrifugated, the samples were separated into aliquots and frozen at -80 °C until analysis. Serum levels of vitamin  D were measured by Radioimmunoassay (RIA), using an immunological test kit (LIAISON® 25 OH Vitamin D TOTAL Assay; DiaSorin, USA), following the manufacturers’ instructions with the PACKARD Copper II E5005 gamma counter analyzer.(USA)
Parathyroid hormone (PTH) was measured by immunoradiometric assay

Ethical consent

Ethics approval: The ethics committee for the faculty of medicine authorized this research, and all procedures were followed in accordance with the laws and regulations that applied.

Consent to participate: Each parent gave their written permission to participate in the research. 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 concerning vitamin D serum levels of mother/ infant (r: 0.466; p < 0.001) in both groups. Table 2

Regarding the biochemical data analyzed; higher levels of PTH were observed in the PTs group. Table 2

We found significant differences in the levels of calcium and phosphorus among the two groups; such did not have clinical
relevance as values were still within the normal range. Table 2

A compartive analysis  was conducted to identify factors associated with vitamin D levels at birth and we found that vitamin D levels were dependent on the mother’s levels .Table 3

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 babaies  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; 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, 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 accompanied by altered 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 serum levels at birth when comparing PTs to term infants, or regarding maternal levels.

We found a positive correlation concerning 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.

CariolouM,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 women should have vitamin D serum levels > 35 ng/mL for optimizing 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 sufficiency and that the vitamin D-PTH relationship must be taken into consideration  and as PTH is a major hormone of bone resorption, and its serum levels are indicator for  secondary hyperparathyroidism and metabolic bone disease in extremely low birth weight neonates and preterms.[44,45]

We found significant differences concerning 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 divided 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 low vitamin D and high PTH levels were associated with high 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 28 days of life. Therefore, PTs admitted to the NICU are at higher risk for vitamin D deficiency. [45] 

The 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 conducted 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 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 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]

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.