Neonatal iron distribution and infection susceptibility in full term, preterm and low birthweight babies in urban Gambia: study protocol for an observational study

Abstract

Background: Neonatal infection is the third largest cause of death in children under five worldwide. Nutritional immunity is the process by which the host innate immune system limits nutrient availability to invading organisms. Iron is an essential micronutrient for both microbial pathogens and their mammalian hosts. Changes in iron availability and distribution have significant effects on pathogen virulence and on the immune response to infection. Our previously published data shows that, during the first 24 hours of life, full-term neonates have reduced overall serum iron. Transferrin saturation decreases rapidly from 45% in cord blood to ~20% by six hours post-delivery. Methods: To study neonatal nutritional immunity and its role in neonatal susceptibility to infection, we will conduct an observational study on 300 full-term normal birth weight (FTB+NBW), 50 preterm normal birth weight (PTB+NBW), 50 preterm low birth weight (PTB+LBW) and 50 full-term low birth weight (FTB+LBW), vaginally-delivered neonates born at Kanifing General Hospital, The Gambia. We will characterize and quantify iron-related nutritional immunity during the early neonatal period and use ex vivo sentinel bacterial growth assays to assess how differences in serum iron affect bacterial growth. Blood samples will be collected from the umbilical cord (arterial and venous) and at serial time points from the neonates over the first week of life. Discussion: Currently, little is known about nutritional immunity in neonates. In this study, we will increase understanding of how nutritional immunity may protect neonates from infection during the first critical days of life by limiting the pathogenicity and virulence of neonatal sepsis causing organisms by reducing the availability of iron. Additionally, we will investigate the hypothesis that this protective mechanism may not be activated in preterm and low birth weight neonates, potentially putting these babies at an enhanced risk of neonatal infection. Trial registration: clinicaltrials.gov ( NCT03353051) 27/11/2017.

Keywords: Hepcidin; Host-Pathogen Interaction; Hypoferremia; Neonates; Nutritional Immunity; Sub-Saharan Africa; The Gambia; Transferrin.

Conflict of interest statement

No competing interests were disclosed.

Copyright: © 2019 Cross JH et al.

Figures

Figure 1.. Main study flow chart of all study procedures and exclusion criteria.

Group A will contain neonates characterised by preterm birth and low birthweight (PTB+LBW); Group B will contain neonates characterised by preterm birth and normal birthweight (PTB+NBW); Group C will contain neonates characterised by full term birth and low birthweight (FTB+LBW); Groups D1, D2 and D3 will all contain babies characterised by full term birth and normal birthweight (FTB+NBW). In this study, preterm is defined α): Father refused, mother refused, family/escort refused, communication not possible or mother with severe disabilities. Exclusion criteria (β): Antibiotics or antimalarials given before delivery (<24 hours), referred to tertiary level health facility, absconded, known HIV-positive, severe pre-eclampsia, receiving TB treatment, antepartum haemorrhage, recent blood transfusion (within the last month), no foetal heartbeat, mother <18 years, refusal, recruited to another study and emergency caesarean section. Exclusion criteria (γ): Recruited to another study on-site, refusal, blood transfusion given in labour, antibiotics or antimalarials given during labour, neonate requires resuscitation (1 min APGAR), neonatal weight <2000g, neonate born breech, neonate born via vacuum delivery, neonate born caesarean section, foetal stillbirth, macerated stillbirth and major congenital malformations. Exclusion criteria (δ): Failed cord blood collection (serum tubes), failed cord blood collection (EDTA), cord blood processed >6 hours, neonate requires resuscitation (10 min APGAR), absconded and route 2B refusal. Exclusion criteria (ε): Mother birth check refusal, father birth check refusal, family escort birth check refusal, mother <18 years, recruited to another study on-site, antibiotics or antimalarials given to mother before delivery (<24 hours), neonate has had surgery, neonates sibling twin was recruited, neonate given antibiotics (other than tetracycline eye ointment), neonate given iron supplementation, neonatal sickness (tone, activity, feeding, heart rate, respiratory rate, abnormal anterior fontanelle), neonatal temperature (<36.5°C or >37.5°C), major congenital malformations (neonate), New Ballard Score (<32 weeks), failed V1 (serum), failed V1 (EDTA), failed V1 (both EDTA and serum), mother V1 bleed refusal, father V1 bleed refusal, and family/escort V1 bleed refusal. Exclusion criteria (ζ): neonatal sickness (tone, activity, feeding, heart rate, respiratory rate, abnormal anterior fontanelle), neonatal temperature (<36.5°C or >37.5°C), neonate has had surgery, neonate given antibiotics (other than tetracycline eye ointment), neonate given iron supplementation, failed V2 bleed, Mother community/V2 bleed refusal, father community/V2 bleed refusal, and family community/V2 bleed refusal.

Figure 2.. NeoInnate Study enrolment route and blood draw design.

Group A contains neonates characterised by preterm birth and low birthweight (PTB+LBW); Group B contains neonates characterised by preterm birth and normal birthweight (PTB+NBW); Group C contains neonates characterised by full term birth and low birthweight (FTB+LBW); Groups D1, D2 and D3 all contain babies characterised by full term birth and normal birthweight (FTB+NBW).

Figure 3.. Estimated power to detect a given difference between Groups A vs D based on simulation using a linear regression model adjusted for baseline for three sample size scenarios.

N1 (Group A=Group B=Group C=50 neonates);N2 (GroupA=GroupB =25 neonates and Group C=50 neonates);N3 (GroupA=GroupB =10 neonates and Group C=50 neonates);N4 (GroupA=Group B=50 neonates and Group C =10 neonates).

Figure 4.. An example of hypothetical scenario for TSAT values between the groups to be compared.

In this example: (i) Time 0 refers to average cord blood levels (ii) Time 6–24 refers to the mean level in the 6–24 hour period after birth. (iii) T1, T2, represent TSAT in 1 and 2 above and ΔT=T2-T1 for full term, normal birthweight (Group D) (iv.) T1’, T2’and ΔT’=T2’-T1’ same as above but for the premature, low birthweight (Group A). Hypothesis: H 0: T2=T2’ vs. H A: T2≠T2’.