Efficacy of zinc supplementation for young infants with clinical severe infection in Tanzania: study protocol for a randomised controlled trial

Abstract

Introduction

Innovative interventions will be essential for countries in sub-Saharan Africa to achieve the 2030 Sustainable Development Goal for child mortality. Infections among young infants, including sepsis, meningitis and pneumonia, continue to cause a large burden of morbidity and mortality in low-income and middle-income countries. Zinc is an essential micronutrient with a well-established role in human health and immune system function, and supplementation may improve survival and treatment outcomes for infants with bacterial infections.

Methods and analysis

We will conduct an individually randomised, quadruple-blind trial of zinc supplementation among 3250 infants 0–59 days old with clinical severe infection (CSI) in Dar es Salaam, Tanzania. Infants with CSI will be randomised to receive either (1) zinc citrate supplementation consisting of 5 mg elemental zinc taken two times per day for 14 days or (2) a matching placebo supplementation taken two times per day for 14 days. Infants will be followed for 90 days postrandomisation. The coprimary outcomes are (1) infant death (all-cause mortality to 90 days) and (2) treatment failure (composite outcome of death during initial hospitalisation, need for additional respiratory support, use of vasoactive medicines or change of antibiotics). Secondary outcomes include important infant health and nutritional outcomes.

Ethics and dissemination

The trial protocol was approved by Harvard T. H. Chan School of Public Health Institutional Review Board, the Muhimbili University of Health and Allied Sciences Institutional Review Board, the National Health Research Ethics Sub-Committee and the Tanzania Medicine and Medical Device Authority. Findings will be disseminated locally, regionally and internationally at scientific conference presentations and as peer-reviewed publications.

Trial registration number

NCT06102044; ClinicalTrials.gov identifier

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Infants with clinical severe infection defined by a clinical presentation of movement only when stimulated, not feeding well on observation, temperature ≥38°C, temperature <35.5°C or severe chest in-drawing are at high risk for mortality and poor treatment outcomes.

• Randomised trials of zinc supplementation in combination with standard antibiotic treatment may improve the survival of infants with clinical severe infection. However, efficacy data are absent in sub-Saharan Africa, and the effect of zinc may vary by aetiology of infections, antibiotic resistance rates, extent or severity of zinc deficiency as well as genetic and other factors that vary by region.

WHAT THIS STUDY ADDS

• This trial will be the first randomised controlled trial of zinc supplementation for infants with clinical severe infection in sub-Saharan Africa.

• The trial will also provide important information on the aetiology of clinical severe infection and treatment outcomes in Tanzania.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Our trial findings will provide evidence for decision-making on global guidelines for the treatment of children with clinical severe infection.

Introduction

Sustainable Development Goal 3 (SDG 3) calls on all countries to achieve a child mortality rate for children under 5 years of age of less than 25 per 1000 live births with a neonatal mortality rate (NMR) of less than 12 per 1000 livebirths by 2030.¹ In 2019, among the 5.3 million global under 5 (U5) child deaths, 2.4 million (46%) were estimated to occur during the neonatal period (first 28 days of life).² The global mortality rate for children 1–59 months of age has declined by approximately 50% since 1990, while the NMR has only declined by 37% during the same period.³ Sub-Saharan Africa has seen the largest disparity between declines in neonatal and postneonatal mortality, as neonatal mortality has only decreased in the region by 28% since 1990.⁴ As a result, the development of simple, effective and low-cost interventions that improve young infant survival will be essential for low- and middle-income countries (LMIC), particularly for countries in sub-Saharan Africa, to reach SDG 3.

Severe bacterial infections among young infants, including sepsis, meningitis and pneumonia, are estimated to cause ~33% of all neonatal deaths.³ The WHO Integrated Management of Childhood Illness (IMCI) algorithm classifies neonates and young infants with clinically suspected sepsis as having ‘Possible Serious Bacterial Infection’ (PSBI).⁵ Clinical severe infection (CSI) is a subset of infants with PSBI that have at least one of five clinical signs associated with particularly high-risk infections: (1) movement only when stimulated, (2) not feeding well on observation, (3) temperature ≥38°C, (4) temperature <35.5°C or (5) severe chest in-drawing.⁶ Antibiotics are recommended for the treatment of young infants with PSBI, including CSI, but even on treatment, the case fatality rate for PSBI can exceed 10% in sub-Saharan Africa.⁷ Zinc is an essential micronutrient with a well-established role in human health and immune system function that is integral to DNA synthesis and for cell growth and differentiation.⁸ Zinc supplementation is known to reduce the duration and severity of diarrhoea episodes, and the WHO and UNICEF have recommended zinc for the treatment of childhood diarrhoea since 2004.⁹ The potential of zinc supplementation in combination with antibiotics for children with sepsis has been evaluated in small randomised controlled trials.10,13 The most recent meta-analysis included data from four trials (including 986 participants) that were all conducted in India and found that zinc supplementation significantly reduced the risk of mortality by 52%, but the uncertainty in this estimate was high, and no individual trial was adequately powered to detect a mortality effect.¹⁴ There is an ongoing randomised trial of zinc for CSI that is being conducted in India and Nepal that will enrol 4140 hospitalised infants with primary outcomes of death during hospitalisation and at 12 weeks of follow-up.¹⁵ No trials of zinc for CSI or PSBI have been conducted in the context of sub-Saharan Africa.

We will conduct a randomised, quadruple-blind, placebo-controlled trial of zinc supplementation among young infants 0–59 days with CSI in Tanzania. All enrolled young infants will receive standard clinical management, including antibiotics, and will be randomised to receive either a 14-day course of two times oral 5 mg elemental zinc citrate tablets (10 mg per day) or a matching placebo regimen. This trial will provide evidence from sub-Saharan Africa to inform global policy decision-making on zinc supplementation for young infants with CSI.

Methods

Study design and setting

We will conduct a randomised, quadruple-blind, placebo-controlled trial of zinc supplementation among 3250 young infants (0–59 days) hospitalised with CSI as defined by IMCI criteria in Dar es Salaam, Tanzania (clinicalTrials.gov identifier: NCT06102044). The trial will be conducted in hospitals with an inpatient neonatal intensive care unit, including Amana Regional Referral Hospital, Temeke Regional Referral Hospital and Mwananyamala Regional Referral Hospital. The trial protocol was developed by collaborators in Tanzania (Muhimbili University of Health and Allied Sciences) and the USA (Boston Children’s Hospital and Harvard T.H. Chan School of Public Health). This trial protocol was written following the Standard Protocol Items: Recommendations for Interventional Trials checklist.

Eligibility criteria and recruitment

Research staff will assess the eligibility criteria for infants who present for care at trial clinics. The trial inclusion criteria are (1) young infants aged 0–59 days; (2) CSI diagnosis as defined by (a) movement only when stimulated, (b) not feeding well on observation, (c) temperature ≥38°C or <35.5°C, (d) severe chest in-drawing; (3) ability to feed enterally; (4) caregiver and participant intend to stay in study area for 90 days of follow-up, (5) provide written informed consent. Exclusion criteria include: (1) prior use of zinc supplements during the current illness, (2) receipt of antibiotics for >24 hours before enrolment, (3) diarrhoea at enrolment, (4) signs suggestive of serious illness/condition that is not CSI, (5) previously enrolled in the trial, (6) enrolled in another research study. Mothers or caregivers will also be asked to provide written consent for the storage of data and blood specimens collected in the trial for future studies.

Interventions

Eligible young infants whose mothers/primary caregivers’ consent for trial enrolment will be randomised to receive one of two regimens: (a) zinc citrate supplementation consisting of 5 mg elemental zinc taken two times per day for 14 days or (b) placebo supplementation consisting of placebo supplements taken two times per day for 14 days. There will be no discernible difference between the zinc and placebo supplements in appearance, taste, smell or any other feature. The trial supplements are manufactured by Vitaquest International (West Caldwell, New Jersey) and were purchased at a discounted rate by the trial team.

Trial study staff will directly administer the trial regimen every 12 hours during the hospitalisation period. The regimen supplements will be dissolved in 2.5 mL of breastmilk or sterile bottled water. The infant will be observed for 30 min after each trial dose. If the infant vomits within 30 min of regimen administration, the infant will be redosed. At the time of hospital discharge, the parent or caregiver will be given the regimen bottle to take home and counselled to give the tablets two times per day until all 14 days are completed. Mothers will be instructed to repeat doses if the infant vomits within 30 min of administration. Study staff will collect the regimen bottles on day 15 of follow-up.

Randomisation and blinding

Participants will be randomised in a 1:1 ratio to the zinc or placebo supplementation groups, stratified by trial clinic. The allocation sequence was generated by non-study staff through computer-generated randomisation lists of participant IDs stratified by trial clinic with block randomisation (random block sizes of 4, 6, 8 and 10). A pharmacist, not involved in the study, will prepare regimen bottles that are labelled with participant IDs using the clinic randomisation lists. During the randomisation visit, research staff will assign participants the next available participant ID, which matches a prelabelled regimen bottle. The randomisation procedures were developed to ensure complete allocation concealment and blinding. The trial is quadruple-blind in that the participants, investigators, outcome assessors and data analysts will be blinded to all participants’ randomisation group.

Concomitant care provided during the trial

All participants will be provided with the standard of care by clinic staff (not study staff) according to the national guidelines for care in Tanzania. Antibiotic treatment protocols for young infants with CSI will be standardised and consistent with the Tanzanian national guidelines.

Data collection

The trial flow diagram is presented in figure 1. At the randomisation visit, study staff will collect anthropometric measurements and administer a standardised questionnaire to collect information on sociodemographic characteristics. In addition, all infants will have blood collected at baseline to perform a blood culture, a complete blood count and to have plasma stored for zinc quantification.

Figure 1. Trial flow diagram.

Once per day during hospitalisation, a study physician will perform a complete physical exam, and study staff will record antibiotics, respiratory support, blood pressure medications given and other treatments administered since the last study visit. The clinic staff will determine the timing of infant discharge from the hospital. At discharge, the study team will record any prescriptions to be taken by the participant at home and measure infant length and weight using standardised procedures. Weight will be measured using an electronic scale, and length will be measured using a length board within 0.1 cm. All measures will be taken in duplicate; however, if the difference between measurements is greater than 0.1 kg or 0.5 cm, then a third measure will be taken. Length-for-age, weight-for-length and weight-for-age z-scores using the WHO Child Growth Standards will be calculated from the means of duplicate weight and length measures; however, in the case that three measurements were taken, the median value will be used for z-score calculations.

All infants will have a clinic visit on day 15 after the expected completion of the 14-day trial regimen. At this visit, a study physician will complete a physical exam, collect information on current medications received and history of rehospitalisation, and study staff will measure infant length and weight and conduct a pill count to assess regimen adherence. Infants will also have 2 mL of blood collected on day 15 for a complete blood count and to have plasma stored for zinc quantification. Phone call visits will then be conducted on days 30, 45, 60 and 75 to assess infant vital status and collect information on rehospitalisation. The trial discharge visit will be completed on day 90. At the trial discharge visit, a study physician will complete a physical exam, collect information on current medications received and history of rehospitalisation, and study staff will measure infant length and weight.

Outcome definitions

The coprimary outcomes of the trial are (1) infant death and (2) treatment failure. Infant death will be defined as all-cause infant mortality to 90 days. Treatment failure will be defined as a composite endpoint including (1) death during initial period of hospitalisation, (2) the need for additional respiratory support (either mechanical ventilation or positive end expiratory pressure support) or (3) the use of vasoactive medicines to support blood pressure or (4) need to change antibiotics during the initial hospitalisation. Our definition of treatment failure is consistent with completed and ongoing trials of zinc for the treatment of CSI in Nepal and India.¹⁵ The secondary trial outcomes and their definitions are presented in table 1.

Table 1. Secondary outcome definitions.

Secondary outcome	Definition	Time period
Duration of initial hospital stay	Days from randomisation to hospital discharge	Randomisation to the date of initial hospitalisation discharge, assessed up to 90 days
Duration of presenting signs of clinical severe infection	Days from randomisation to the absence of any sign of clinical severe infection high body temperature≥38°C low body temperature<35.5°C severe chest indrawing movement only on stimulation stopped feeding well	Randomisation to day 90
Diarrhoea during initial hospital admission	Clinical diagnosis based on three or more loose or watery stools in the past 24 hours	Randomisation to the date of initial hospitalisation discharge, assessed up to 90 days
Vomiting related to regimen dosing during initial hospital admission	Vomiting observed by study staff within 30 min of regimen dosing	Randomisation to day 15
Infant re-hospitalisation	Infant admitted and stayed overnight in health facility after being discharged from initial hospitalisation	Discharge to day 90
Presence of any sign of possible severe bacterial infection	Presence of possible severe bacterial infection:  high body temperature ≥38°C  low body temperature <35.5°C  severe chest indrawing  movement only on stimulation or no movement at all  stopped feeding well or unable to feed at all  convulsions	Day 15 and day 90 (separate analyses)
Diarrhoea	Maternal report of three or more loose or watery stools in the past 24 hours	Day 15 and day 90 (separate analyses)
Infant length-for-age z-score	Infant length-for-age z-score by WHO Child Growth Standards	Day 15 and day 90 (separate analyses)
Infant weight-for-age z-score	Infant weight-for-age z-score by WHO Child Growth Standards	Day 15 and day 90 (separate analyses)
Infant weight-for-length z-score	Infant weight-for-length z-score by WHO Child Growth Standard	Day 15 and day 90 (separate analyses)
Plasma zinc concentration	Infant plasma zinc concentration	Day 15

Sample size

Power calculations were based on enrolment of 3250 infants with 1:1 randomisation to the zinc and placebo regimen (1625 per arm) with a nominal type 1 error rate (alpha) of 0.05 and a 95% retention rate. Table 2 presents the statistical power to detect the relative risk (RR) of the outcome in those randomised to zinc compared with placebo, varying the cumulative incidence of the two primary endpoints in the placebo arm. The trial will have excellent power to detect an RR of 0.80 on mortality, even if the cumulative incidence is as low as 17.5% in the placebo group. The expected effect size of a 20% reduction of mortality is also reasonable; the most recent meta-analysis on the efficacy of zinc for sepsis found a RR of 0.48 (95% CI 0.25 to 0.94).¹⁴ We also expect the composite outcome of treatment failure to have a similar or greater incidence, as it includes mortality within the facility.

Table 2. Statistical power for trial primary outcomes with 3250 participants, 1:1 randomisation, a nominal type 1 error rate (alpha) of 0.05, and a 5% loss to follow-up rate.

90-day mortality—primary outcome #1
Cumulative incidence in placebo group	Relative risk zinc vs placebo
	RR=0.70	RR=0.75	RR=0.80
15%	96%	87%	68%
17.5%	98%	92%	76%
20.0%	99%	96%	83%
Treatment failure—primary outcome #2
Cumulative incidence in placebo group	Relative risk zinc vs placebo
	RR=0.70	RR=0.75	RR=0.80
17.5%	98%	92%	76%
20.0%	99%	96%	83%
22.5%	99%	98%	88%

Statistical analysis

An intent-to-treat approach with a complete case analysis will be the primary analytic strategy for all outcomes. For the coprimary outcomes of infant death and treatment failure, log-binomial models, including a fixed effect for study clinic to account for stratified randomisation, will be used to estimate the RRs, 95% CIs and p values. If log-binomial models do not converge, log-Poisson models will be used. For secondary outcomes, non-repeatable binomial outcomes will similarly be analysed with log-binomial models that include a fixed effect for the study clinic will be used. For repeatable binomial outcomes, generalised estimating equations with the log link, binomial distribution, exchangeable correlation matrix and robust estimators of variance were used to assess the RRs. For continuous secondary outcomes, linear regression models with a fixed effect for study clinic will be used to account for the stratified randomisation scheme to produce mean differences. We will conduct sensitivity analyses that adjust for baseline factors that showed some degree of imbalance between treatment groups based on a p<0.20. We will also conduct a time-to-event sensitivity analysis for the coprimary outcomes using Cox proportional hazard models.

Oversight and monitoring

The trial will be reviewed by a Data and Safety Monitoring Board (DSMB) that will meet every 6 months to review study progress and safety data during the conduct of the trial. The prespecified interim efficacy analysis for the two primary endpoints, all-cause mortality and treatment failure, will be performed at one time point when half of the participants have completed the study follow-up (90 days). The efficacy interim analysis will include RR estimates and if the p value for a difference is overwhelming (as evidenced by a p value less than 0.001), the DSMB may request unblinding and a recommendation of stopping may be considered. The DSMB will monitor all severe adverse events (SAEs) at each meeting and review events by masked treatment group. If the comparison of an SAE varies significantly between arms at a p value of 0.05 or below, the study will be evaluated for safety concerns and considered for stopping. The trial will also be reviewed by an external study monitor who will also conduct regular audits throughout the duration of the trial.

Patient and public involvement

Patients and the public were not involved in the trial design.

Ethics and dissemination

The trial protocol was approved by Harvard T. H. Chan School of Public Health Institutional Review Board (Ref. No. IRB23-0138), the Muhimbili University of Health and Allied Sciences Institutional Review Board (MUHAS-REC-02-2024-2019), the National Health Research Ethics Sub-Committee (NatHREC) (Ref. No. NIMR/HQ/R.8c/Vol. I/2831) and the Tanzania Medicine and Medical Device Authority (TMDA) (Ref No. BC.69/96/99/01). All participants’ mothers or caregivers will provide written informed consent. The trial will be overseen by a DSMB that will meet every 6 months during the conduct of the trial.

We will disseminate the primary and secondary findings of our trial through publications in academic journals. We will also present selected results at conferences, research seminars and scientific meetings. We will also disseminate our findings to government and non-government partners.

Discussion

Our trial will provide causal evidence on the efficacy of zinc supplementation adjunct to antibiotic treatment for CSI Tanzanian young infants. This trial evidence from sub-Saharan Africa is intended to be used with data from South Asia in global decision-making on the use of zinc for CSI. Our trial is innovative, efficient and potentially groundbreaking as we evaluate whether an inexpensive and readily available nutrient supplement can improve young infant survival and treatment outcomes. Evidence from this randomised trial will contribute to evidence on potential interventions that improve young infant survival to support Tanzania and other LMIC to reach the SDG for child mortality by 2030.