Trial setting {9}
The trial is conducted in 14 primary and secondary health facilities in three districts of the northern province of Sierra Leone (Bombali, Tonkolili, and Port Loko). This region is characterised by a tropical climate, with a rainy season from May to October and a dry season from November to April. The Northern region has one of the highest U5 malaria prevalence (40%) and infant mortality rates in the country (102 infants per 1,000 live births) (20).
Eligibility criteria {10}
Participants must meet the following criteria to be eligible for the trial:
- Caregivers agree to participate and have signed the informed consent form (ICF)
- Aged ≤10 weeks at enrolment visit
- Being eligible for receiving pentavalent-1 vaccine as per the routine EPI Sierra Leone guidelines
- Body weight ≥2.5 kg at enrolment visit
- Permanent resident in the trial area
- Without known allergies to or contraindications to macrolides
- Without known allergies to or contraindications to SP
- Caregivers agree to complete the child’s EPI scheme at the trial recruitment health facilities
The primary exclusion criteria are as listed below:
- Aged >10 weeks old at enrolment visit
- Body weight <2.5kg at enrolment visit
- Residing outside the trial area or planning to move out in 12 months from enrolment
- Known history of allergy or contraindications to macrolides and/or formulation excipients
- Known history of allergy or contraindications to SP and/or formulation excipients
- With signs of any acute illness that prevents the child from being vaccinated at the time of enrolment
- Diagnosed with chronic hepatic, cardiac or renal disease
- Enrolled in any other interventional trial
Who will take informed consent? {26a}
All caregivers or legal representative guardians of the eligible children are provided with trial information. A signed or thumb-printed ICF is obtained before any trial intervention can take place. If the caretaker is below the legal adult age of 18 years, they are required to assent for child participation in the trial, in addition to a legal guardian adult providing consent according to Sierra Leone national ethics recommendations.
Additional consent provisions for collection and use of participant data and biological specimens {26b}
For the subset of children who are asked to provide biological samples, additional oral consent is sought before sample collection is performed.
Interventions
Explanation for the choice of comparators {6b}
A comparator with no therapeutic effect (placebo) and similar appearance, taste, and smell is used in this trial. Both AZI and comparator packaging are similar, and reconstitution and administration follow the same procedures. This will minimize any potential bias that may inadvertently influence the trial outcomes.
Intervention description {11a} Azithromycin or placebo administration
Recruited participants are assigned a trial number and randomised into either the AZI or placebo arms. All enrolled infants received a single dose of oral AZI/placebo powder for oral suspension (supplied by PLIVA Croatia Ltd.) after randomisation. The dosage is based on body weight (volume of reconstituted 200mg/5ml suspension equivalent to 20mg/kg). AZI/placebo administration is under close supervision. Infants are observed for 30 minutes after administration. Infant vomiting within 30 minutes of administration is given a repeated full dose. No additional dose of AZI/placebo is administered to any child who vomits the second time. Trial drug safety and tolerability are assessed within the first seven days of administration through home visits. The subsequent doses of AZI/placebo are administered at 9 months of age alongside MRV 1 and yellow fever immunisations, and at 15 months of age alongside MRV 2 immunisation.
PMC-SP administration
PMC-SP is administered at 10 and 14 weeks and at 6, 9, 12, and 15 months of age alongside routine EPI. The dosage of PMC-SP is given as an oral dispersible tablet (250mg/12.5mg) according to body weight (Table 1). Children older than 1 year of age receive two tablets, regardless of their body weight. All children receiving PMC-SP are observed for 30 minutes after administration. Infant vomiting within 30 minutes of administration are given a repeated full dose. No additional dose of PMC-SP is administered to any child who vomits for the second time.
Table 1. Sulfadoxine-pyrimethamine (SP) dosage by body weight and age.
|
Weight (Kg)
|
Treatment
(1 tablet =250mg/12.5mg)
|
|
<5*
|
½ tablet
|
|
5-10*
|
1 tablet
|
|
>10**
|
2 tablets
|
*and aged<1 year of age, **or aged>1 year of age
Criteria for discontinuing or modifying allocated interventions {11b}
Study treatment can be discontinued for the following reasons: a) requirement for prohibited concomitant medications or other contraindications to AZI; b) occurrence of an adverse event requiring discontinuation of AZI; c) request by participants’ caretakers to terminate study treatment; and d) clinical reasons believed to be life-threatening by the physician in charge. Additionally, participants’ caregivers or legal representatives have the full right to end their participation in the trial at any time.
Strategies to improve adherence to interventions {11c}
The trial drugs are administered at each scheduled visit, and caretakers are advised to follow the Sierra Leone EPI calendar. The trial team works closely with Sierra Leone health authorities to ensure that all required interventions are available at both recruitment health facilities and outreach points. In addition, safety contact numbers have been established, so caregivers can contact the trial team at any time to proactively manage and address adverse events. Finally, community social mobilisers engage families and community leaders to address potential barriers to adherence and develop tailored solutions.
Relevant concomitant care permitted or prohibited during the trial {11d}
Caretakers are asked not to administer any macrolide antibiotics to their children unless they are critically needed with no alternative drugs available.
Provisions for post-trial care {30}
Trial insurance provides coverage for trial-related injuries or deaths to participants. However, there is no provision for post-trial care.
Outcomes {12}
The primary outcome is the rate of all-cause mortality at 18 months of age.
The secondary outcomes include:
a) Cause-specific mortality rate and malaria related mortality at 18 months of age
b) Incidence of all-cause hospital admissions
c) Incidence of all-cause outpatient attendances
d) Frequency and severity of drug adverse reactions
e) Prevalence of SP resistance in U5
f) Prevalence of macrolide resistance in nasopharyngeal isolates
g) Prevalence of macrolide resistance in the gut bacteria
h) Proportion of children with protective antibody responses to specific routine EPI (measles, rubella and yellow fever)
Mortality surveillance
Both passive and active case-detection surveillance systems have been established at trial health facilities and communities to capture mortality cases. The mortality outcome will be determined using a Demographic Surveillance System (DSS) established in the catchment area of each study health facility. Verbal autopsies are performed to determine the cause of death of all participants who died in the communities, and the possible cause of death is determined by a panel of experts using the available clinical record information. A verbal autopsy questionnaire adapted from the WHO 2016 verbal autopsy instrument and a computerised method (Inter-VA) are used to interpret the cause of death (21).
Assessment of the effect of additional doses of PMC-SP on malaria risk
Two non-contemporary cohorts have been established to compare malaria risk between children receiving only three doses of PMC-SP (n=250, cohort of non-trial participants) and those receiving six doses of PMC-SP (n=500, cohort of trial participants) in the same trial area. Participants are enrolled at the time of the EPI visit during routine pentavalent 2 vaccination and the first PMC-SP administration, and both cohorts are followed up to 24 months of age. The trial participants cohort are recruited from the same health facilities and in the same season as the non-trial cohort to minimise differences in malaria exposure. Active malaria detection with malaria RDT and haemoglobin assessment using the HemoCue Hb301 system take place four–six weeks after MRV2 visits for both the non-trial and trial cohorts. Finger-prick blood on filter papers as dry blood spots (DBS) are collected during this visit for the trial cohort participants to detect sub-clinical malaria parasitaemia and to assess potential P. falciparum resistance to AZI. Passive malaria surveillance at each EPI contact and other unscheduled visits to the health facilities are used to assess the health status including malaria incidences in these cohort children.
Assessment of parasite resistance to SP
Two health facility-based cross-sectional studies are conducted before and after the trial implementation to measure the prevalence of molecular markers associated with SP resistance among symptomatic U5 children diagnosed with malaria residing in the trial catchment areas.
Finger-prick blood samples as DBS are collected from the recruited children onto filter papers to determine the presence of P. falciparum dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) gene mutations.
Assessment of parasite resistance to macrolides
The trial is assessing the prevalence of AZI-resistant bacterial organisms (Streptococcus pneumoniae, and Staphylococcus aureus) at recruitment and three months after the first and last doses of AZI/placebo administration in nasopharyngeal swabs collected from a subset of participants. Nasopharyngeal specimens are collected on duplicate paediatric calcium alginate swabs. The first nasopharyngeal swab of the participant is immediately placed into 1 ml skim milk-tryptone-glucose-glycerol medium and the second nasopharyngeal specimen is stored in DNA/RNA Shield (Zymo Research) as a backup sample and, if required, to analyse the microbiome and resistome of the nasopharyngeal samples using a shot-gun approach (22). Nasopharyngeal samples are transported on ice and stored at −80°C for batch processing. To assess the effect on gut bacterial composition and macrolide resistance, rectal samples are collected from a subset of trial children at recruitment and three months after the first and last doses of AZI/placebo administration. Rectal swabs samples are collected with calcium alginate swabs, stored in the DNA/RNA Shield (Zymo Research), transported on ice and stored at -20ºc.
Assessment of interactions of AZI with routine EPI immunisations
A prospective study is being conducted to assess the effect of AZI on the serological response to measles, rubella, and yellow fever vaccines through EPI. The objective of this study is to compare the proportion of infants who achieve protective titers against measles, rubella, and yellow fever vaccine between the AZI and placebo arm, three months post MRV1 and yellow fever vaccination. A subset of infants receiving MRV1 and yellow fever vaccines at 9-months old during the EPI visit are assessed. To ensure an equal number of participants from both trial arms (AZI and placebo), 1/3 of the target sample size is being recruited from each trial treatment allocation letter (A to F) to obtain the target sample size in each treatment group, regardless of the three letters corresponding to each one. A venous blood sample (1 ml) is being collected from each child before MRV1, and yellow fever vaccination are administered at 9 months of age. Another blood sample is collected 3 months post-vaccination at the 12-month EPI visit.
Participant timeline {13}
Table 2. Participant timeline.
|
trial/EPI visit (age)
|
Penta 1 Enrolment
(≤ 10w)
|
Penta 2 (10w)
|
Penta 3
(14w)
|
Vit. A
(6m)
|
MRV1
(9m)
|
Vit. A
(12m)
|
MRV2
(15m)
|
18
m
|
Household visits
|
Unscheduled visits
|
|
trial procedure
|
|
Inclusion/ exclusion criteria check a
|
x
|
|
|
|
|
|
|
|
|
|
|
Written informed consent a
|
x
|
|
|
|
|
|
|
|
|
|
|
Demographics a
|
x
|
|
|
|
|
|
|
|
|
|
|
Previous doses Adverse events
|
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
|
Weight a
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
|
|
|
|
Length
|
|
x
|
|
x
|
x
|
x
|
x
|
|
|
|
|
AZI/Placebo administration b
|
x
|
|
|
|
x
|
|
x
|
|
|
|
|
PMC-SP administration c
|
|
x
|
x
|
x
|
x
|
x
|
x
|
|
|
|
|
Nasopharyngeal swab d, e
|
x
|
|
x
|
|
|
|
|
x
|
|
|
|
Rectal samples e
|
x
|
|
x
|
|
|
|
|
x
|
|
|
|
Blood samples f
|
|
|
|
|
x
|
x
|
x
|
|
|
|
|
RDT/Haemoglobin blood spots g
|
|
|
|
|
|
|
x
|
|
|
|
|
Drug tolerability assessment h
|
x
|
|
|
|
x
|
|
x
|
x
|
x
|
x
|
|
Mortality surveillance i
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
x
|
a. Based on infants age (≤ 10 weeks) and eligible for receiving Pentavalent 1 vaccine
b. AZI: Azithromycin; AZI/placebo depending on assigned groups
c. PMC-SP : Perennial malaria chemoprevention for malaria in infants with SP
d. Sample collection from a subgroup of randomly selected trial participants for the assessment of resistance to macrolides in the nasopharyngeal sample at recruitment and three months after the first and last dose of AZI/placebo administration
e. For the assessment of resistance to macrolides in gut flora, rectal samples is being collected from relatives of the randomly selected subgroup of trial children at recruitment and three months after the first and last dose of AZI/placebo administration
f. Blood sample is being taken from a subgroup of participants immediately before and three months after yellow fever and/or MRV administration for the assessment of AZI interaction with EPI.
g. To assess the effect of additional IPTi-SP doses on the prevalence of malaria and anaemia
h. Only after AZI/Placebo administration
i. To measure the impact of AZI on child survival
Sample size {14}
A sample size of 20,560 infants (10,280 per arm) is estimated to achieve 18.5%-20.0% reduction in all-cause mortality at 18 months of age in infants receiving AZI at 6 weeks, 9 months, and 15 months of age compared to infants receiving placebo at each of the three contacts, with 80% statistical power and alpha error of 0.05 (23). This calculation is based on a baseline infant mortality rate of 41-48 per 1,000 live births at 18 months of age in the control group, estimated using a mortality rate of 83 per 1000 adjusted for recruitment between 6 and 10 weeks of age, assuming a 20% loss to follow-up (3).
The sample sizes for the cohort, SP resistance, macrolide resistance, and AZI/vaccine interaction studies are 250 per arm, 300, 936, and 333 per arm, respectively. Details of the sample size calculations for each substudy are provided in Annex 1.
Recruitment {15}
Newborns are identified during their first EPI visits immediately after birth. Caregivers are contacted once their children turn six weeks old to be reminded to visit the health facility for their children to receive pentavalent 1 vaccine visit and enrolment in the trial. Caregivers of children born and identified in the communities within the trial catchment areas are also encouraged to visit any of the trial health facilities to be considered for enrolment.
Assignment of interventions: allocation
Sequence generation {16a}
Participants are allocated to trial arms centrally by the trial sponsor (ISGlobal) in a 1:1 ratio in 343 blocks of 60. Each trial number is linked to the allocated treatment groups. The treatment packs are masked by a single letter (A to F).
Concealment mechanism {16b}
The trial drugs (AZI and placebo) are identical in colour, flavour, and packaging. Trial number allocation for each trial participant is concealed in opaque sealed envelopes and are only opened after the child meets all eligibility criteria.
Implementation {16c}
The sponsor statistician generates the trial allocation sequence. The identification numbers are managed by the data-management office. The first identification number is assigned to the first recruited child, and the subsequent numbers are assigned to subsequent recruited children in a chronological sequence by trial nurses at randomisation. Trial nurses administer the assigned drug (AZI or placebo) to the participants following standard operating procedures (SOPs).
Assignment of interventions: Blinding
Who will be blinded {17a}
All site personnel, outcome assessors, data analysts, and participants’ caregivers remain blinded throughout the trial.
Procedure for unblinding if needed {17b}
The coding system for randomization includes a mechanism for rapid unblinding in case of a medical emergency, which does not allow undetectable breaks of the blind. If a medical emergency arises where the investigator needs to break the treatment code immediately, he/she must seek the sponsor's designated representative approval before requesting unblinding information. A case report form is used to capture all required information.
Data collection and management
Plans for assessment and collection of outcomes {18a}
Data is collected in an electronic case report form using Research Electronic Data Capture (REDCap) and uploaded regularly to a secure cloud-based server (24). The trial identification card provided to the participants contains a QR code linking their records to the database. The source documents are designed to capture scheduled and unscheduled trial visits, demographic surveillance events, and medical records captured through a morbidity and mortality surveillance system.
The demographic surveillance system records the basic demographic event information required to capture all deaths in the trial children occurring at both the community and health facility levels. The passive-case detection surveillance system captures the clinical data of all trial children who report being sick at health facilities.
Plans to promote participant retention and complete follow-up {18b}
Caretakers of trial participants are informed of the importance of compliance with all trial procedures and requirements during recruitment and subsequent follow-up visits. The caretakers of trial participants who miss any of the EPI visits are reminded through phone calls and home visits about the importance of compliance. Participant re considered loss to follow-up if they fail to attend a scheduled visit and is unreachable for their regular follow-up visits or end-of-trial follow-up visit at 18 months of age.
Data management {19}
A real-time quality control system ensures that errors and inconsistent data entries that do not meet the software in the built validation logic rules, including mandatory value ranges and fields, are flagged and checked. All paper-based trial information is stored in fire-proof cabinets with a controlled access system for the entire retention period. Trial data management, in collaboration with the quality control and clinic teams, generates periodic reports, reviews, and resolves all queries.
Confidentiality {27}
The database system has been designed to protect confidentiality (sensitive data is automatically encrypted) and integrity of the data, including authorisation, authentication, auditing, and availability features to safeguard data access and usage. The database can only be accessed by trial-authorised personnel. The trial sponsor has no access to any trial participant’s personal identifiers. Publication of trial results will not include any participant’s personal data in line with the standard International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use-Guideline for Good Clinical Practice (ICH-GCP) principles.
Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
To assess P. falciparum resistance to SP, DNA from the collected DBS is being extracted and quantified. Dhfr and dhps genotypes are being determined using multiplex amplicon sequencing. Dhfr mutations at codons 51, 59, and 108 and dhps mutations at codons 437, 540, 581, and 613 are being analysed. To assess macrolide resistance, NPS samples in STGG medium are being inoculated onto Columbia nalidixic acid (CNA) agar and incubated. Presumptive S. aureus and S. pneumoniae colonies are sub-cultured on selective media and identified using MALDI-TOF and optochin (Becton Dickinson Gmb) (15µg at 14 mm breakpoint), respectively. The minimum inhibitory concentrations of confirmed S. pneumoniae and S. aureus isolates are determined using AZI stripe (0.016-256 µg/ml) for the E-test according to the European Committee on Antimicrobial Susceptibility Testing Clinical Breakpoint (25). DNA is being extracted and quantified from rectal swab samples. The V3-V4 variable regions from the 16S rRNA gene (amplicon size expected ~460 bp) are PCR-amplified using the MiSeq® System platform from Illumina (2x 300pb paired-ended). In addition, a subset of the samples are evaluated using shotgun faecal metagenomics sequencing (26). To assess the interactions of AZI with measles, yellow fever, and rubella vaccines, blood samples are collected in a serum separator microtainer and undergoes centrifugation. All sera samples are frozen at -20 oC until analysis. Antibody titres against measles and yellow fever are measured using a microneutralisation test. To this aim, sera samples are subjected to serial dilutions before mixing them with a standardised concentration of virus. This solution is plated on virus-susceptible cells, and the antibody effects are measured (27). Antibody titres against rubella are measured using an enzyme-linked immunoassay (ELISA) test (27). Infants in the AZI intervention group are compared with those in the placebo group. Neutralising antibody titre concentrations of 120 mIU/mL are considered the best surrogate markers for measles protection: >1:10 for yellow fever and anti-rubella IgG titers of > 10 IU/mL for rubella. Infants whose antibody concentrations before vaccination suggest previous exposure to the outcome of interest (measles, rubella, and yellow fever) will be excluded from statistical analyses.
Statistical methods
Statistical methods for primary and secondary outcomes {20a}
Data from the primary objective (rate of all-cause mortality at 18 months of age) will be estimated using Cox regression models, for both the crude and adjusted risk of death associated with the treatment arm. The protective effect (PE) will be estimated from the Hazard Ratio (HR) as PE =100*(1-HR)%. Regarding secondary outcomes, continuous variables will be described as mean, standard deviation, median, minimum, and maximum. Categorical variables will be described by the number and percentage of subjects in each category. The Chi-square test (or Fisher’s exact test) and t-test will be used as appropriate to verify the homogeneity of both groups. The incidence of clinical malaria, overall admissions, and outpatient attendance will be estimated as the number of episodes over time at risk. Time at risk will be estimated as the time from the start of follow-up (dose 1) until the end of follow-up (18 months of age) or withdrawal due to censoring or death, whichever occurs first. To avoid counting twice the same clinical malaria episode, trial participants will not contribute to the denominator or numerator during an arbitrary period of 28 days after the episode of clinical malaria is defined. A maximum of one episode of admission or outpatient visit will be counted per day. The total number of events will be compared between groups using negative binomial regression with random effects to consider between-child heterogeneity. The comparison will be expressed as the incidence rate ratio. The analysis will be performed using STATA version 17.
Interim analyses {21b}
Not applicable. There are no interim analyses planned.
Methods for additional analyses (e.g. subgroup analyses) {20b}
Not applicable. There are no additional methods for analyses planned.
Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}
The primary efficacy analysis will be based on an intention-to-treat approach, using all randomised participants with outcome data. If any statistical method is required to account for missing data in the secondary outcomes, multiple imputations will be used.
Plans to give access to the full protocol, participant level-data and statistical code {31c}
The datasets derived from the trial can be made available upon reasonable request.
Oversight and monitoring
Composition of the coordinating centre and trial steering committee {5d}
This is a single centre trial coordinated by ISGlobal in collaboration with the College of Medicine and Allied Health Sciences (COMAHS), University of Sierra Leone, and the Ministry of Health, Sierra Leone. The trial day-to-day support is provided by
International Principal Investigator: design and develop the trial protocol and lead training for personnel.
Local Principal Investigator: oversees the day-to-day clinical trial activities on site and ensures full compliance with ICH-GCP principles, ensuring that trial documents are submitted to the relevant authorities.
Co-investigators: provide support on data collection, management and reporting.
The trial coordinator coordinates day-to-day activities of the trial to ensure that recruitment targets are met and full compliance with the trial protocol requirements.
Data manager: develops data collection tools. Ensures that reports are generated for routine quality checks and query resolution.
Statistician: calculates the trial sample size, develops the statistical analysis plan, and performs the statistical analyses.
Sub-Investigators: ensure that recruited children are well monitored, evaluated, and any serious adverse events properly managed.
Pharmacists: manage and handle drugs storage, inventory and dispensing processes.
Clinical trial monitors: perform monitoring visits to ensure that the site fully complies with the approved protocol and safeguards participant safety in line with the ICH-GCP guidelines.
The trial also has a safety monitoring team that reviews all serious adverse event cases and provides timely feedback to the site team on a regular basis. The trial has no steering committee group.
Composition of the data monitoring committee, its role and reporting structure {21a}
The trial has a Data and Safety Monitoring Board (DSMB) in place. This is an independent committee consisting of experts in infectious diseases, paediatrics, epidemiology, pharmacology, biostatistics, and other relevant disciplines. The DSMB reviews and analyses all clinical safety data collected during the trial and assesses unexpected or serious adverse events that may occur throughout the trial. It is also responsible for providing advice on safety-related issues before trial initiation, during, and close out.
Adverse event reporting and harms {22}
The on-site principal investigator is responsible for recording, managing, and reporting the adverse events that occurred during the trial. The severity of any adverse event is being assessed using the Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Paediatric Adverse Events (28). All adverse events and SAEs are being recorded in the REDCap database application system and reported to all relevant authorities in a timely manner. SAEs are defined as any AE observed in a trial that meets at least one of the following criteria: i) in death, ii) life-threatening, iii) requires inpatient hospitalisation or prolongation of existing hospitalisation, iv) results in persistent or significant disability/incapacity, or v) is considered a medically important event by the trial physician (29). The severity, causality, and outcome of each SAE is be thoroughly assessed and recorded in REDCap. If a fatal event occurs outside a health facility, verbal autopsy is performed to determine the cause of death following standard trial procedures (21).
Frequency and plans for auditing trial conduct {23}
Trial monitoring is conducted by an external contract research organisation (CRO), Pharmalys Ltd. The CRO ensures that the trial is implemented in line with all the applicable requirements. The CRO has access to participant's personal data and other trial records. It is responsible for conducting site initiation visits, periodic interim monitoring visits, and site close-out visits, based on the approved monitoring plan. Audit and regulatory inspection visits to the trial site can be initiated at any time by national or international health authorities.
Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}
The DSMB, regulatory authorities, and Sierra Leone Ethics Committee are informed by the investigators of all major changes to the protocol, informed consent, and other trial-related key documents that may adversely affect the safety of the participants or the conduct of the trial.
Dissemination plans {31a}
A project communication plan is being developed to ensure timely, accurate, and effective communication between partners and key stakeholders. The plan outlines the responsibilities for data management, sharing, and publication strategies. After concluding the trial analysis of all relevant data, the results will be made available to all the partners and key stakeholders. The trial results will be shared and disseminated to the scientific community and beyond. Dissemination of information to the scientific community will be done through reports, scientific publications in international journals, and presentations at scientific and other appropriate forums.