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Bacillus Calmette-Guerin (BCG) vaccination has been reported to protect neonates from non-tuberculous pathogens, but no biological mechanism to explain such effects is known. We hypothesised that BCG produces broad-spectrum anti-microbial protection via a hepcidin-mediated hypoferraemia, limiting iron availability for pathogens.To test this we conducted a trial in 120 Gambian neonates comparing iron status in the first 5-days of life after allocation to: (1) All routine vaccinations at birth (BCG/Oral Polio Vaccine (OPV)/Hepatitis B Vaccine (HBV)); (2) BCG delayed until after the study period (at day 5); and (3) All routine vaccinations delayed until after the study period.Vaccine regime at birth did not significantly impact on any measured parameter of iron metabolism. However, the ability to detect an effect of BCG on iron metabolism may have been limited by short follow-up time and high activation of the inflammatory-iron axis in the study population.
80 healthy Gambian neonates were randomly allocated to receive BCG (Danish Strain 1331, Batch 11023B, 0.05 ml intra-dermally into the left deltoid) either at birth, or after completion of study procedures at five days old. All other routine immunisations (Oral Polio Vaccine (OPV)) and Hepatitis B Vaccine (HBV) were given at birth as normal. A data manager not directly involved in the study, conducted randomisation using Microsoft Access, upon delivery of an eligible infant. Blocked randomisation using blocks of six with a 1:1 allocation ratio was used. Due to concerns regarding the potential confounding influence of OPV and HBV at birth, a third non-randomised group of 40 infants was subsequently recruited and received all vaccinations after completion of study procedures at five days of age. Recruitment ran from May 2013 until February 2014, with the first two, randomised groups, recruited during both rainy and dry seasons, and the third non-randomised group recruited during the dry season. All participants had a 2 ml baseline venous blood sample taken within 24 h of delivery, prior to receipt of any vaccinations, and a further 2 ml venous blood sample taken either 24–48 or 72–96 h post-intervention. Blood was collected directly into microtainers (Becton–Dickson: 0.5 ml collected into EDTA containing tubes, 1.5 ml into lithium–heparin containing tubes) from the dorsum of the hand. Full blood counts were assessed from EDTA blood using the automated Medonic analyser. Lithium–heparinised blood was centrifuged for 4 min at 3600 g within 4 h of collection and the plasma stored at −70 ˚C until analysis. Iron parameters were measured using the automated Cobas Integra 400 plus (Roche Diagnostics). Plasma hepcidin was measured in duplicate, using a 1:20 dilution by competitive ELISA (Bachem-25, USA) with detection range 0.02–25 ng/ml. Plasma IL-6 was measured in duplicate using a 1:2 dilution by competitive ELISA (BD OptEIA, Oxford, UK), with detection range 0.49–250 pg/ml. Samples with readings outside the linear portion of the curve were re-run at alternative dilutions. Values below the limit of detection were imputed using limit of detection/√2. Any samples with an intra-assay co-efficient of variance >15% were re-analysed. Demographic, birth details and anthropometry were collected at enrolment. Due to the rural nature of the study site, all births were vaginal. Deliveries and follow-up visits were conducted at the participant’s home. Full informed consent was obtained from mothers antenatally by a trained midwife. Inclusion criteria were (1) Consenting mother (2) Residence within the study area. Exclusion criteria were (1) Infant weighing <2000 g (2) Maternal HIV or TB (3) TB contact in the home (4) complicated delivery (5) major congenital anomaly (6) infant unwell as judged by a doctor or a midwife. The Consort flow diagram for the study can be found as supplementary material. Clinical investigators and mothers were not blinded to intervention allocation due to lack of feasibility (BCG produces a visible reaction) and for safety, so that any mothers would be aware of the vaccination status of the child. Laboratory investigators were blinded to intervention allocation, with assays conducted by anonymous study number. Data were analysed using Stata Version 11.0. Categorical variables were compared using the chi-squared test and continuous variables by one-way ANOVA. Hepcidin and IL-6 results were not normally distributed and were log-transformed prior to comparison. Intervention allocation code was not broken until the data were cleaned and locked. As this study was a small proof-of-principal trial, with short follow-up and no clinical endpoints, no data safety monitoring board was appointed. Safety data were monitored in real time by clinical investigators who were not blinded to intervention allocation. There was no significant difference in incidence of serious adverse events by intervention allocation group (see Table 1). Population characteristics by intervention group. Ethical approval was obtained from the joint Gambia Government/MRC Unit The Gambia ethics committee (Ref: SCC1325) and the London School of Hygiene and Tropical Medicine ethics committee (Ref: 012-045). This trial was conducted according to the principles of the Declaration of Helsinki.
