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Confidential: For Review OnlyEffect of dose reduction of supplemental zinc for childhood diarrhea: study protocol for a double-masked, randomized,
controlled trial in India and Tanzania
Journal: BMJ Paediatrics Open
Manuscript ID bmjpo-2019-000460
Article Type: Protocol
Date Submitted by the Author: 13-Feb-2019
Complete List of Authors: Somji, Sarah; Muhimbili University of Health and Allied Sciences, PediatricsDhingra, Pratibha; Centre for Public Health Kinetics (CPHK)Dhingra, Usha; Centre for Public Health Kinetics (CPHK)Dutta, Arup; Centre for Public Health Kinetics (CPHK)Devi, Prabhabati; Centre for Public Health Kinetics (CPHK)Kumar, Jitendra; Centre for Public Health Kinetics (CPHK)Deb, Saikat; Centre for Public Health Kinetics (CPHK)Semwal, Om Prakash; Centre for Public Health Kinetics (CPHK)Sazawal, Sunil; Centre for Public Health Kinetics (CPHK)Manji, Karim; Muhimbili University of Health and Allied Sciences, PediatricsKisenge, Roderik; Muhimbili University of Health and Allied Sciences, PediatricsBakari, Mohamed; Muhimbili University of Health and Allied Sciences, PediatricsAboud, Said; Muhimbili University of Health and Allied Sciences, PediatricsLiu, Enju; Children's Hospital BostonSudfeld, Chris; Harvard University T H Chan School of Public HealthDuggan, Christopher; Children's Hospital BostonAshorn, Per; Tampereen yliopistoBahl, Rajiv; Organisation mondiale de la SanteSimon, Jonathon; Organisation mondiale de la Sante,
Keywords: General Paediatrics, Paediatric Practice, Pharmacology, Tropical Inf Dis
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1 Effect of dose reduction of supplemental zinc for childhood diarrhea: study protocol for
2 a double-masked, randomized, controlled trial in India and Tanzania
3
4 Sarah S Somji* ([email protected])1, Pratibha Dhingra*([email protected])2
5 and the Zinc Therapeutic Dose Trial (ZTDT) Team**
6 * Joint First Authors
7 ** ZTDT Team by alphabetical order of sites:
8 India (Center for Public Health Kinetics, New Delhi):
9 Usha Dhingra ([email protected]); Arup Dutta ([email protected]);
10 Prabhabati Devi ([email protected]); Jitendra Kumar
11 ([email protected]); Saikat Deb ([email protected]); Om Prakash Semwal
12 ([email protected]) and Sunil Sazawal ([email protected])
13 Tanzania (Muhimbili University of Health and Allied Sciences, Dar es Salaam and Boston
14 Children’s Hospital, Boston):
15 Karim Manji ([email protected]); Rodrick Kisenge ([email protected]);Mohamed
16 Bakari ([email protected]); Said Aboud ([email protected]); Enju Liu
17 ([email protected]); Christopher Sudfeld ([email protected]) and
18 Christopher Duggan ([email protected]).
19 WHO (Maternal Newborn Child and Adolescent Health Department):
20 Per Ashorn ([email protected]), Rajiv Bahl ([email protected]) and Jonathon Simon
21 ([email protected]).
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22 Correspondence to: Dr. Jonathon Simon, Department of Maternal, Newborn, Child and
23 Adolescent Health, World Health Organization, 20, avenue Appia, 1211 Geneva 27,
24 Switzerland at [email protected]
25 Affiliations:
26 1. MUHAS-Harvard Projects, Muhimbili University of Health and Allied Sciences,
27 Dar-es-Salaam, Tanzania
28 2. Center for Public Health Kinetics, New Delhi, India
29 3. World Health Organization, Geneva, Switzerland
30 Address for reprints: World Health Organization, 20, avenue Appia, 1211 Geneva 27,
31 Switzerland
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32 Abstract
33 Background: Diarrhea associated mortality and morbidity are highest in infants and young children in low- and
34 middle- income countries (LMICs). Zinc supplementation during acute diarrhea has been shown to reduce the
35 duration of illness and the risk of persistent diarrhea. However, vomiting with zinc supplementation is a
36 common side effect that may interfere with compliance and programmatic scale-up, and may be related to the
37 dose prescribed.
38 Methods/design: The Zinc Therapeutic Dose Trial (ZTDT) is a two-center (Tanzania and India), three-arm
39 randomized, double-blind controlled non-inferiority trial. Children 6-59 months of age with acute diarrhea are
40 eligible to participate. Enrolled children (1500 per arm; 4500 total) will be randomly allocated to receive 5, 10
41 or 20 mg of zinc sulfate daily for 14 days and will be followed up for 60 days after enrollment. All children will
42 receive WHO-UNICEF IMCI standard of care (oral or intravenous rehydration and zinc as indicated and feeding
43 advice). The primary efficacy outcomes of the trial are the percentage of subjects with diarrhea duration more
44 than 5 days, the mean total number of loose or watery stools after enrollment and the proportion of children
45 vomiting within 30 minutes of zinc administration.
46 Discussion: The ZTDT trial will determine the optimal dose of therapeutic zinc supplements for treatment of
47 acute diarrhea in children aged 6-59 months in two LMICs. The results of the trial are likely to be generalizable
48 to childhood acute diarrhea in similar resource-limited settings and may influence global policy about zinc
49 supplementation dosage during acute diarrhea.
50 Trial Registration: ClinicalTrials.gov identifier NCT03078842. Registered on March 13, 2017. Protocol
51 Version/Date: Zinc Protocol Version 4.0/ 19/09/2016.
52 Keywords: Zinc supplementation, Under-five, Children, Optimal dose, Randomized-controlled trial,
53 Acute diarrhea
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54 Key Messages5556 Therapeutic zinc supplementation using 20 mg zinc became a component of WHO - UNICEF 5758 standard diarrhoea case management in 2004. 5960 New research evidence had shown zinc reduced the duration and severity of the episode, and 6162 lowered the incidence of diarrhoea in the following 2-3 months.6364 This study evaluates whether lower doses of zinc (10mg or 5 mg) are non-inferior in clinical 6566 efficacy and reduce the common side effect of vomiting. 6768 If vomiting is less frequent, caregiver acceptability and willingness to treat the child may 6970 improve leading to greater utilization and better health outcomes. 7172 Background:
73 Zinc is a vital micronutrient essential for protein synthesis, cell growth and differentiation,
74 immune function, and intestinal transport of water and electrolytes [1]. Zinc is also important
75 for normal growth and development of children both with and without diarrhea [2]. Zinc
76 deficiency is associated with an increased risk of gastrointestinal infections, altered structure
77 and function of the gastrointestinal tract, and impaired immune function [3-5]. Dietary
78 deficiency of zinc is especially common in low-income countries because of a low dietary
79 intake of zinc-rich foods (including foods of animal origin) or low bioavailability due to the
80 presence of phytates often found in cereal-based diets [1].
81 The benefits of zinc supplementation in the management of diarrhea have been observed in
82 several studies. Patel et al. [6] noted in a systematic review and meta-analysis an approximate
83 20% reduction in the mean duration of acute diarrhea with zinc supplementation. Lazzerini
84 and Wanzira [7], summarizing data from 2581 children from 9 trials, noted that children older
85 than 6 months receiving zinc supplements had a shorter diarrhea duration by about half a day
86 (mean difference -11.46 hours, 95% CI -19.72 to -3.19). A reduction in diarrhea that
87 continues up to day 7 (risk ratio 0.73, 95% CI 0.61 and 0.88) was also noted in this study.
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88 Moreover, the study indicated that zinc supplements in children with persistent diarrhea
89 (defined as diarrhea for more than 14 days) also may shorten average diarrhea duration by 16
90 hours (mean difference -15.84 hours, 95% CI -25.43 to -6.24). As a result of these and other
91 trials, since 2005 WHO/UNICEF have recommended routine zinc supplementation (10 mg
92 daily for infants below 6 months of age; 20 mg daily for children older than 6 months) for 10-
93 14 days [8].
94 These doses, however, were not chosen after dose-ranging studies, and substantially exceed
95 the recommended dietary allowances (RDA) for zinc in infancy and early childhood (2-5
96 mg/day) [9]. High doses of zinc can cause epigastric pain and lethargy, and its metallic taste
97 can also induce vomiting. Vomiting with zinc supplements is a common side effect noted in
98 many published trials. A meta-analysis by Lukacik et al (2008) [3] summarized the findings
99 for acute and persistent diarrhea from a total of 22 zinc intervention trials, using a range of
100 zinc dosing (5 mg to 45 mg/day). In 11 acute diarrhea trials (n= 4438), the proportion of
101 participants who vomited after the initial dose was significantly higher with zinc use than
102 with placebo (12.7% vs. 7.6%; RR: 1.55; 95% CI: 1.30–1.84; P ≤0.001). According to
103 Lazzerini and Wanzira’s review [7], the risk of vomiting increased with zinc supplementation
104 in both children older than age 6 months (risk ratio 1.57, 95% CI 1.32 to 1.86; 2605 children,
105 6 trials) as well as children younger than 6 months of age (risk ratio 1.54, 95% CI 1.05 to
106 2.24; 1334 children, 2 trials). Other studies have also reported that therapeutic zinc
107 supplementation during diarrhea causes higher rates of vomiting [10-12].
108 Thus, although substantial evidence supports the efficacy of zinc supplementation during
109 episodes of diarrhea, vomiting is significantly more common among supplemented children.
110 Lower doses of zinc, provided they are equally effective, might have the advantage of
111 reducing side effects, increasing compliance and programmatic roll-out. If the zinc dose for
112 diarrhea treatment and zinc deficiency can be uniform, then the national essential drug lists
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113 can be simplified. Therefore, there exists an important rationale to identify the dose of zinc
114 supplementation associated with both efficacy and an improved safety profile. We present the
115 protocol for a two-center, randomized, double-blind controlled trial of three doses of zinc
116 conducted in Tanzania and India. The overall goals of the study are to evaluate the non-
117 inferiority, safety and acceptability of the two lower zinc doses (5 and 10 mg/day) versus
118 standard zinc dose (20 mg/day).
119 Methods
120 Study design
121 ZTDT is an individually randomized, parallel group, double-blind, controlled trial of three
122 doses of supplemental zinc conducted among 4500 children ages 6-59 months in India and
123 Tanzania (ClinicalTrials.gov identifier NCT03078842).
124 The trial protocol was developed by collaborators at WHO, Department of Maternal
125 Newborn, Child and Adolescent Health (Geneva, Switzerland) together with teams in New
126 Delhi, India (Centre for Public Health Kinetics), Dar es Salaam, Tanzania (Muhimbili
127 University of Health and Allied Sciences) and Boston, USA (Boston Children’s Hospital and
128 Harvard TH Chan School of Public Health). The ZTDT protocol diagram is presented in
129 Additional file 1. The schedule of trial enrollment, interventions, and assessments is
130 presented in Additional file 2. The first participant was enrolled in the trial in January 2017 in
131 India and March 2017 in Tanzania and follow-up data collection will continue through
132 approximately June 2019. This paper follows the Standard Protocol Items: Recommendations
133 for Interventional trials (SPIRIT) checklist (see Additional file 3).
134 The study population will include 4500 children aged 6-59 months with diarrhea of less than
135 72 hours duration. Enrolled children will be followed up for 60 days after randomization. The
136 participant will be followed up closely for the first 15 days to record post intervention
137 morbidity outcomes and to ensure compliance with study regimen. Study participants will be
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138 assessed again at thirty (30), forty-five (45) and sixty (60) days post enrollment to estimate
139 impact on longer-term morbidity outcomes.
140 Patient and Public Involvement
141 This research was done without patient involvement. Patients were not invited to comment
142 on the study design and were not consulted to develop patient relevant outcomes or interpret
143 the results. Patients were not invited to contribute to the writing or editing of this document
144 for readability or accuracy.
145 Primary and secondary objectives
146 The overall aim of ZTDT is to identify the optimal dose of zinc in children under 5 years with
147 acute diarrhea.
148 The primary aims of the study are: 1) to assess whether lower doses of zinc (5 or 10 mg/day)
149 are at least as effective as the standard dose of zinc (20 mg/day) in children 6-59 months, in
150 terms of burden of illness (percentage of subjects with diarrhea lasting more than 5 days;
151 mean number of loose or watery stools during follow-up) and 2) to evaluate the improved
152 effectiveness by reduction in the risk of vomiting in the 30 minutes after receipt of the
153 supplement in low dose supplemental zinc (5 or 10 mg/day) in comparison to standard dose
154 of zinc (20 mg/day).
155 Secondary aims of the study are: 1) to explore whether lower doses of zinc have any impact
156 on post- acute illness morbidity outcomes assessed at 30, 45 and 60 days, 2) to explore
157 whether duration of treatment, estimated by mother’s reports and collection of used zinc
158 blister packs, has any impact on the primary outcomes, 3) to determine the acceptability of
159 low dose supplemental zinc compared to standard zinc dose based on an acceptability scale
160 and 4) to determine whether the different doses of zinc are associated with differential plasma
161 zinc levels at days 3, 7, 15, 21 and 30 after randomization.
162 Participants/interventions and outcomes
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163 Study setting
164 The study recruitment sites are in South East Asia (SEA) and Sub-Saharan Africa (SSA). In
165 SEA the study recruitment sites are located in Sangam Vihar, a resettlement colony on
166 outskirts of South Delhi and Harsh Vihar, a semi-urban locality in North East District, Delhi,
167 India. In SSA recruitment sites are at health centers in Temeke District Hospital, Mbagala
168 Round Table and Mbagala Rangi Tatu, Dar es Salaam, Tanzania. Both the sites will be
169 recruiting from outpatient health facilities.
170 Eligibility
171 Study participants will be eligible for enrollment in the study if they meet the following
172 inclusion criteria: 1) males and female children between 6 to 59 completed months of age, 2)
173 acute diarrhea duration of less than 72 hours at the time of screening (defined as three or
174 more loose or watery stools) or dysentery (defined as visible blood in the stool), 3) likely to
175 stay within the study area for the next 2 months and 4) written informed consent is obtained
176 from caretaker.
177 Children will be excluded if they: 1) have severe acute malnutrition [weight for length/height
178 (WHZ) Z score of <-3 or edema], 2) have severe dehydration that cannot be corrected within
179 4 to 6 hours, 3) have signs of severe pneumonia (characterized by presence of either chest in-
180 drawing and either of the following; convulsions or unconscious or vomiting everything [13],
181 sepsis, rapid diagnostic test (RDT)-confirmed malaria or other severe illness, 4) have
182 previously or currently been enrolled in the study, 5) are currently enrolled in another trial,
183 6) are not intending to remain in study area for the duration of the study, 7) have a caregiver
184 who declines participation in the study, 8) have other child currently enrolled in the study in
185 the same household and 9) have used zinc supplements during the three days preceding study
186 enrolment.
187 Sample size
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188 We will enroll 4500 children (1500 per study arm) in the trial. Table 1 presents sample size
189 assumptions and calculations for each of the three primary outcomes. The total sample size
190 for the trial was selected to have at least 90% power for each of the primary outcomes. The
191 primary outcomes (mean number of loose or watery stools post-randomization and
192 percentage of children with diarrhea for more than 5 days) are both based on the non-
193 inferiority hypotheses, while the primary outcome of vomiting post-dose assumes a
194 superiority hypothesis. We assumed 1:1:1 randomization between the three treatment arms, a
195 nominal Type I error rate (alpha) of 0.05 and 5% loss to follow-up rate.
196
197
198
199
200 Table 1 Sample size calculations for the primary trial outcomes
201Outcome Value in
standard dose (20 mg/d) group
Expected value in lower dose groups
Non-inferiority margin
Power, Significance level
Sample size per arm (with ~5% attrition)
Duration of diarrhea, mean (SD)
3.0 (2.7) days 3.0 (2.7) daysLower dose equally effective
0.3 days 90%, 5% 1460
Percentage of children with diarrhea >5 days
16% 16%Lower dose equally effective
+4% 90%, 5% 1500
Total number of loose or watery stools during follow up, mean (SD)
10 (9) stools 10 (9) stoolsLower dose equally effective
+2 stools 90%, 5% 1460
Percentage of children with
20% 15%Lower dose
Not applicable
90%, 5% 1340
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vomiting during follow up
safer because of superiority hypothesis for this outcome
202203204 Interventions
205 Children are randomized to receive one of the three zinc sulfate regimens (each taken once
206 daily for 14 days): 1) 5 mg; 2) 10 mg; or 3) 20 mg. The zinc tablet (all 3 regimens)
207 ingredients include aerosil, maize starch, ethylvanillin (a vanilla flavor), aspartame,
208 magnesium stearate vegetal and vivapur. The three study tablets have identical appearance,
209 taste and smell. The supplement is a dispersible tablet that dissolves in water or breast-milk in
210 about 20 seconds. The caretakers are instructed to dissolve the tablet in 5–10 mL of water or
211 breast milk immediately before administration. Children receive zinc supplementation for 14
212 days. The first dose of study drug is given at the health facility in which the child is enrolled.
213 All three doses of the regimen are packaged identically; 10 tablets in 1 blister, 2 blisters in 1
214 pack (extra doses are provided for redosing in case of vomiting). Each pack is pre-labelled
215 with participant ID. Study regimen is manufactured by Laboratoires Pharmaceutiques Rodael
216 S.A.S, France and shipped to WHO for randomization and labelling, before shipment to
217 recruitment sites (as described below in Randomization, allocation, concealment and
218 masking).
219 Adherence assessment
220 Adherence to the study regimen is optimized by close follow up: either in person
221 (clinic/home visits) or telephone contact on days 3, 5, 7, 10 and 15. Adherence to the regimen
222 is measured by counting the numbers of unused tablets during each follow-up visit. Study
223 workers/ research assistants physically count the tablets during home or clinic visits. We give
224 the mother/caregiver a small booklet to record every successful administration as well as any
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225 failed attempts to administer the intervention. Study worker reviews the diary card on each
226 visit and collects filled diary card on day 15 visit.
227 Standard of care
228 All enrolled children receive standard of care for diarrhea management per WHO/UNICEF,
229 Tanzanian, and Indian Ministry of Health guidelines. This includes standardized assessment
230 of hydration status, rehydration and maintenance fluids with intravenous or oral rehydration
231 solutions (ORS) as indicated, recommendation for increased fluid intake and continued
232 feeding at home, and instructions on when to seek follow-up care. Children with known
233 Human Immunodeficiency Virus (HIV), receive care and treatment appropriate for the child
234 and referral to the Care and Treatment Centre nearest to the child’s home.
235 Randomization, allocation, concealment and masking
236 Trial arm allocation was performed according to a computer-generated randomization list
237 (generated off-site by a non-study statistician) of 4500 individuals, stratified by country and
238 child age (>6 - <24 months versus >24 - <60 months). A statistician will hold the
239 randomization list codes until completion of primary analyses or until requested by the Data
240 and Safety Monitoring Board (DSMB). Within each stratum, subjects are randomly assigned
241 equally in permuted blocks of variable size to receive one of the three doses of zinc. The
242 purpose of the stratification is to have equal distribution of younger and older children among
243 all three treatment groups. An independent pharmacy team prepared the zinc regimen in
244 Geneva by labeling blister packs with participant IDs based on the randomization list. Labels
245 were printed at WHO, Geneva, and enrolled children were assigned sequential serial
246 numbers. These pre-labeled regimens were shipped to the sites in Tanzania and India. The
247 packing and physical appearance of the three study regimes are identical. In addition, the
248 three doses of the regimen are similar in taste, appearance and smell. As a result, the research
249 teams, study staff as well as caregivers are fully blinded to treatment arm. Zinc formulations
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250 are serially numbered according to the randomization codes. The code is kept in a password
251 protected spreadsheet file by WHO personnel and is not available to the investigators until
252 the end of the study. This randomization list was converted into unique sequential serial
253 numbers for each enrolled child at each site. This code will only be broken if the DSMB
254 requests this information for a participant [should they suspect a relationship of a suspected
255 Severe Adverse Event (SAE) to the study drug] or for a study arm (in their interim analyses).
256 Data collection, management and analysis
257 Screening and randomization
258 Both sites will be recruiting from community clinics. Surveillance systems are set up at
259 clinics to identify all children presenting with diarrhea. Trained study staff stationed at each
260 health facility are responsible for identification of children who are potentially eligible. The
261 study team is trained in good clinical practice (GCP) as well as confidentiality and research
262 ethics.
263 Written informed consent is sought from the caretaker of children meeting the eligibility
264 criteria. A child can only be enrolled once into the study. The informed consent process
265 includes telling the caretaker that they are free to withdraw from the study at any time, and
266 that participation is entirely voluntary.
267 Following consent, each participant will be assigned a unique subject identification number.
268 A card detailing the subject ID and the health personnel responsible for enrollment will be
269 provided to the caregiver of enrolled children. The patient locator form /subject master log
270 will be the only link between the subject ID and the participant’s name. This information will
271 be stored together with the consent in a locked file cabinet and an online database accessible
272 to only the Project Manager and required study staff for the purpose of patient tracking.
273 A baseline assessment, including a detailed physical examination (record of vital signs,
274 axillary temperature, respiratory rate, pulse/rate, hydration and nutritional status), will be
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275 performed by a research physician at the time of screening for enrollment. The child is
276 evaluated for any Integrated Management of Childhood Illness (IMCI) general danger signs
277 (unable/ able to drink or breastfeed, convulsions, vomiting, unconscious, stiff neck) and
278 evidence of dehydration or pneumonia, as well as information on recent medical history
279 (duration of diarrhea, bloody stool/dysentery, recent fever). Vital signs (temperature/heart
280 rate/respiratory rate) in dehydrated child are recorded after the child is rehydrated and
281 stabilized and remaining clinical assessment/ screening is completed thereafter.
282 Anthropometry measurements will be taken after rehydration and stabilization as required,
283 before completing the screening for enrolling the participant. Weight will be measured using
284 an electronic scale with a sensitivity of ±10 g by two independent observers; length (for
285 children less than 24 months old) or height (for children 24 months or older) which will be
286 measured using a length board to within 0.1 cm. Mid-upper arm circumference (MUAC) will
287 be measured using non-stretchable tape to the nearest 0.1 cm. All anthropometric measures
288 will be recorded in duplicate (India) and triplicate (Tanzania). Once these measurements and
289 other eligibility criteria are met, the subject will get enrolled.
290 Baseline socio-demographic information and vaccination history
291 Parents of enrolled subjects will be interviewed at study clinic or at home for the baseline
292 socio-demographic characteristics viz. parent's literacy level and occupation, type of house,
293 possession of assets and possible exposures (recent antibiotic use, travel history, water use
294 and filtration and sanitation), breastfeeding and vaccination history and information is
295 recorded in the case record forms.
296 Follow-up visits
297 Scheduled follow-up visits begin from the third day of the enrolment and are generated daily
298 via an automated visit generation module of the data management software. All children
299 enrolled at the study clinic are to be followed up in the clinic or at home by a trained field
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300 worker on day 3, 5, 7, 10, 15, 30, 45, and 60 (Additional file 1 and 2). In India, study staff
301 will visit the household of enrolled children for all follow up visitations. If the child is not
302 available at home, a phone call will be made to follow-up the child. In Tanzania, a participant
303 will be followed up at home or clinic on Days 3, 7, 15, 30 and 60 and by phone on days 5, 10
304 and 45. Children still having diarrhea after Day 15 are treated according to standard
305 management guidelines [8]. At each visitation during active follow up period, a study worker
306 asks the mother or other caregiver about the following and records the responses in a
307 predesigned form/data entry module:
308 1. Ongoing symptoms of diarrheal illness, including the number and consistency of stools
309 2. Vomiting episodes including any episode within 30 minutes following zinc administration.
310 3. Adherence with zinc study regimen ascertained via mothers’ recall and/or reported use of
311 zinc tablets by the child in the Diarrhea Diary Card and cross-checked by determining the
312 number of unused tablets.
313 4. Any adverse events, including hospital admissions and deaths of study participants.
314 5. Criteria for returning to clinic (e.g. lethargy, poor oral intake, dehydration).
315 6. Any other inter-current illness
316 7. Reported use of antibiotics and IV/other fluids
317 At day 30, 45, and 60 post-enrolment, the study participants are assessed either at the study
318 clinic, via telephone or with a home visit for presence of any illness, diarrhea, fever, cough,
319 or fast or difficult breathing within last 24 hours and any time in past 2 weeks. On the Day 60
320 study completion clinic visit, in addition to morbidity assessment, anthropometric
321 measurements will be taken for each participant using the same procedure as mentioned in
322 the baseline assessment.
323 Children not available on the visitation day will be visited the next day; if they are still not
324 available, they are followed-up until found or deemed lost to follow-up. Caregivers are asked
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325 to contact the study physicians if they feel that their children are sick between the visits.
326 Children sick on the day of visitation will be referred to the study clinic for a detailed
327 morbidity assessment as well as treatment by the study physician. During each visit a record
328 of all the medicines and IV fluids given, information on number and consistency of stools and
329 data on adherence is recorded in the predesigned forms. Close monitoring will be
330 accomplished through repeated follow-ups during the 14-day regimen period. Every effort
331 will be made to track and successfully follow-up all randomized children.
332 Study withdrawal or discontinuation
333 The participants have the right to withdraw from the study at any time. The subjects will be
334 considered withdrawn from the trial in case of death or lost to follow-up. However, every
335 effort will be made to track and successfully follow-up all enrolled children. The information
336 on withdrawn or discontinued participants will be recorded through a withdrawal slip by field
337 worker and clinic supervisor.
338 Blood sampling and laboratory investigations
339 To allow the measurement of the potential differential impact of the varying zinc doses on
340 plasma zinc levels during the follow-up period, each study participant will provide two blood
341 samples. One third of the study participants will have blood draws at Days 1 and 15, one
342 third at Days 3 and 21, and the remaining one third on Days 7 and 30 post-enrolment for
343 assessment of plasma zinc levels.
344 Blood drawing will follow the protocols of the International Zinc Nutrition Consultative
345 Group (IZINCG). These include use of powder-free gloves, zinc-free syringes and butterfly,
346 needles and trace element free blood tubes [14]. Plasma is separated at 15 minutes after blood
347 collection and aliquots are transferred into trace element-free Eppendorf plastic tubes for
348 temporary storage at -20°C and then shipped to central lab for final storage at -80°C in India.
349 In Tanzania, plasma samples are stored in site refrigerators at 2-8oC and then shipped to
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350 central laboratory storage at -80oC by the end of the day. Plasma zinc concentrations will be
351 measured by atomic absorption spectroscopy (AAS-400 Perkin Elmer, USA).
352 Quality assurance
353 The persons with at least a secondary school degree will be recruited for data collection team.
354 The following quality assurance checks will be done regularly to ensure highest level of data
355 integrity and quality:
356 ● An extensive 1-3 weeks training session for data collectors to ensure the inter-data
357 collector reliability is above 95%.
358 ● Pilot field data testing is monitored closely by the research scientist(s) responsible for
359 quality assurance.
360 ● Real time electronic data capture will ensure data validation such as range, logical checks
361 and data integrity.
362 ● Interim aggregate data analysis will be carried out periodically for data pattern, heaping,
363 missing values, etc. Based on the feedback, effective measures will be taken to
364 eliminate/minimize the errors.
365 For overall visit supervision, quality control (QC) supervisors will perform quality checks by
366 visiting at least 10% of the households, re-interviewing 5% respondents and cross-checking
367 this data with concerned field worker. QC supervisors are primarily responsible for overall
368 field related project activities (data quality and integration at all levels, resolving community
369 issues), and training. Computer randomized list of enrolled children will be used by quality
370 control team for random household visits.
371 The QC team works in close coordination with the investigative team and provides regular
372 feedback on quality issues. Quarterly meetings between quality control team and senior
373 investigative team are scheduled to discuss overall quality issues and related corrective
374 measures to be undertaken.
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375 Data management
376 Data are collected in real-time using either tablets or laptop computers. Each site uses an in-
377 house designed data management software with user friendly graphical user interface (GUI)
378 for data collection. Generic case report forms (CRFs) and data dictionary are used to maintain
379 data consistency across sites. In-built range, logical and skipping pattern will ensure data
380 quality and integrity.
381 The database is password protected with restricted access. Each user is assigned a unique user
382 identifier to access the software and an audit trial is maintained for database operations. The
383 centralized server located at each site synchronizes the data on daily basis. Data are shared
384 monthly with WHO data quality monitoring team who is responsible for data quality checks
385 and provide feedback to sites. They generate query forms which are returned to the study
386 sites for verification.
387 Data security and storage
388 A backup of the data is kept in the server at the WHO protected by a specific password and
389 accessible to only authorized users. An additional backup of the data is kept in a password
390 protected external hard drive at a site away from the WHO at the recruiting site central
391 offices.
392 Record retention and archival
393 All the study documents including participant’s source data and documents will be archived
394 by the study sites after the completion of the study, till the time WHO informs in writing to
395 the study sites that they no longer need to maintain the study documents.
396 Outcome measures
397 The primary efficacy outcomes are 1) the percentage of enrolled children who have duration
398 of diarrhea of more than 5 days, 2) the mean number of loose or watery stools after enrolment
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399 and 3) the percentage of children vomiting within 30 minutes of administration of the zinc
400 tablet.
401 Secondary outcomes of the trial include: i) proportion of children experiencing serious
402 adverse events (life-threatening or hospitalization; ii) proportion with intermediate duration
403 of diarrhea (diarrhea continuing beyond three days)); iii) Proportion of guardians with
404 positive attitude towards treatment; iv) treatment adherence; v) mean serum plasma zinc
405 concentration at days 1, 3, 7, 15, 21 and 30; vi) illness symptoms between day 15 and 60 after
406 the treatment 2-week period prevalence and number of days with diarrhea, fever or
407 respiratory symptoms. vii) 60-day change in height/length, weight, MUAC, height/length-for-
408 age z-score, weight-for-length/height z-score, weight-for-age z-score, MUAC z-score,
409 stunting, wasting, underweight; viii) adherence to regimen; ix) study dropouts; x) maternal
410 report of ease in supplement administration; xi) maternal report of child acceptability; xii)
411 maternal recommendation of use for other children; xiii) maternal report of change in child
412 skin condition, child appetite, activity levels, mood, and diarrhea severity.
413 Statistical analysis plan
414 All primary analyses of non-inferiority tests will be conducted on the principle of intention-
415 to-treat (ITT). All non-inferiority margins will be defined a priori. We will also conduct
416 sensitivity analyses using a per-protocol (PP) approach [15]. The analysis population for the
417 ITT approach will include all randomized patients with assessable data, regardless of whether
418 the participant is known to have received a full 14-day dose of zinc or not. The analysis
419 population for the PP approach will include study participants who are documented to have
420 taken zinc supplementation for the first 5 days after randomization regardless if they vomited
421 the dose. The PP definition was selected due to the primary outcome being diarrhea duration
422 >5 days. Descriptive statistics will be used to summarize background demographic and
423 baseline clinical characteristics by trial arm.
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424 Analysis of Primary Outcomes
425 Primary Outcome 1: Number (%) of subjects with diarrhea episodes >5 days. The last day of
426 diarrhea will be defined as the day in which there is passage of the last liquid or semi-liquid
427 stool prior to two diarrhea-free days (days when less than 3 loose stools / day). The duration
428 of diarrhea will be defined as the number of days between randomization (not the reported
429 day of onset of diarrhea symptoms before randomization) and the last day with diarrhea.
430 Children who are lost to follow-up, die, or withdraw before 5 days will be excluded from this
431 analysis. For the primary analysis, we have set the threshold for non-inferiority of lower
432 doses of zinc (10 mg/d or 5 mg/d) as compared to the standard 20 mg/d at less than 4% in
433 absolute risk (risk difference) compared to the standard dose in the proportion of subjects
434 with prolonged diarrhea. The proportions of participants with a prolonged diarrhea episode
435 (defined as >5 days) will be calculated in all treatment arms, and the risk differences (and
436 their 95% CI’s) between the lower doses (5 mg/d and 10 mg/d) and the standard dose (20 mg)
437 will be estimated. A Cochran–Mantel–Haenszel (CMH) approach will be used to take
438 stratifying randomization factors, e.g. country and age groups, into account. If the upper limit
439 of the 95% CI for the risk difference is less than 4%, we will conclude non-inferiority for one
440 or both lower dose arms. We will also examine the relative risk (RR) of prolonged diarrhea
441 episodes in both lower dose arms compared to the standard dose arm using relative risk
442 regression which accounts for factors included in the stratified randomization. In secondary
443 analyses, Cox proportional hazard models will be used to estimate hazard ratios and their
444 95% confidence intervals for recovery from diarrhea in each of the lower dose arms
445 compared to the standard dose arm. The time to recovery is defined as the duration of
446 diarrhea as noted above.
447 Primary Outcome 2: Total number of loose or watery stools after enrolment. An Analysis of
448 Covariance (ANCOVA) approach will be used to estimate the mean differences and their
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449 95% CI in the total number of loose or watery stools after enrollment for each lower dose arm
450 compared to the standard dose arm, while accounting for the stratified randomization design.
451 The non-inferiority margin was set at 2 stools, therefore if the upper limit of the 95% CI is <2
452 stools, we will reject the null hypothesis that the lower dose zinc is inferior to the standard
453 dose in term of reducing the total number of loose or watery stools.
454 Primary Outcome 3: Vomiting within 30 minutes after administration of the trial regimen.
455 We hypothesize that the lower dose of zinc will be associated with a reduced risk of vomiting
456 and therefore a superiority test will be used. Vomiting within 30 minutes of each daily zinc
457 dose is repeatedly measured during the 14-day administration period. Generalized estimating
458 equations (GEE) with the log link, binomial variance, and exchangeable correlation matrix
459 will be used to assess the relative risk of vomiting among the three treatment arms. The
460 exchangeable working covariance matrix and robust estimators of the variances will be used
461 to construct confidence intervals. The robust estimators are consistent estimators of the
462 variances even if the working correlation matrix is mis-specified. Stratified randomization
463 factors will be considered in the model. A secondary analysis concerning severe vomiting
464 (defined as vomiting requiring hospitalization or vomiting requiring intravenous fluids) will
465 also be performed in similar fashions. We will also examine the proportion of children ever
466 vomiting between the treatment arms and examine whether the relative risk of vomiting
467 differs by follow-up day.
468 Analysis of change in plasma zinc and other secondary outcomes
469 Due to the sampling design of participants having paired blood sampled on three schedules
470 (days 1 and 15; days 3 and 21; days 7 and 30), we will assess the effect of the randomized
471 zinc regimen at both the population- and individual-level. For the analysis of population-level
472 zinc, we will use a mixed model approach to compare patterns of plasma zinc concentrations
473 over time between the randomized treatment arms. The main exposure variables in the model
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474 will include treatment arm, study day, and an interaction term between treatment arm and
475 study day. In case there is not a linear relationship between study day and plasma zinc
476 concentrations, restricted cubic splines will be used to examine the non-linear relationship
477 between these two variables. The interaction term between zinc dose and study day in the
478 model measures whether the trajectory of plasma zinc differs by zinc supplementation over
479 the full period. If the overall test for this interaction term is statistically significant, group
480 means at each assessment day will then be compared using least-square means with the
481 Tukey-Kramer adjustment. To be consistent with our stratified randomization study design,
482 stratification factors will be included in the model.
483 For all other secondary outcomes (see clinicaltrial.gov NCT03078842), we will use
484 approaches similar to the primary analyses, including an ANCOVA approach for non-
485 repeated continuous outcomes, relative risk regression for non-repeated binomial outcomes,
486 and GEE or mixed effect models for repeated longitudinal outcomes.
487 Sensitivity analyses and effect modification of treatment effect
488 Although randomization is designed such that, on average, treatment groups will be balanced
489 with respect to all baseline prognostic factors, this may not be true in all cases. As a result,
490 we will re-assess treatment effects as observed in the original analysis after adjusting for
491 factors that are potentially imbalanced across randomized arms. If imbalance occurs, we will
492 compare and present the results for both unadjusted and adjusted analyses.
493 In addition, the study was not designed to detect effect modification of the treatment effect
494 among subgroups of children. As a result, we acknowledge that we are likely underpowered
495 to detect differences in the magnitude of a treatment effect among subgroups unless there is
496 strong effect modification. The potential baseline effect modifiers we plan to explore include:
497 study site (India/Tanzania), child age (<12 months vs. >12 months), child sex (male vs.
498 female), prior receipt of rotavirus vaccine (Yes vs. No), maternal HIV status (Yes vs. No),
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499 dysentery at enrollment (Yes vs. No), baseline dehydration status (Yes vs. No), baseline
500 axillary temperature (<38°C vs. >38°C), baseline respiratory rate, recent antibiotic use (Yes
501 vs. No), inter-current illness (cough, or difficulty breathing, or earache), current breastfeeding
502 status (Yes vs. No), and prior breastfeeding status (Yes vs. No). Additional variables that we
503 will test for effect modification will include child anthropometric measures (length/height for
504 age, weight for age, and weight for length/height Z scores, and MUAC), family
505 socioeconomic status (<median wealth index, >median wealth index), maternal education,
506 household water quality (improved vs. unimproved), household sanitation quality (improved
507 vs. unimproved), baseline plasma zinc levels (<median, > median), and trial regimen
508 adherence (<median, >median). Factor analysis will be used to derive wealth index from
509 family possession variables. UNICEF/WHO JMP definitions will be used to clarify improved
510 vs. unimproved for the household water quality and sanitation [16].
511 Monitoring
512 Data and Safety Monitoring Board (DSMB)
513 A Data Safety and Monitoring Board has been established to monitor severe adverse events
514 (SAEs) and to review the statistical analysis plan and associated stopping rules for benefit,
515 futility, or harm determined using O’Brien-Fleming stopping boundaries. The DSMB
516 includes 5 members with expertise in clinical trials, statistics, child mortality assessment,
517 pediatric care in resource limited settings, or practical experience from each study country.
518 The DSMB meets electronically or in person at least every 6 months. Severe adverse events
519 (SAEs) related to study participation are monitored in real-time, are summarized and reported
520 to study investigators, WHO and relevant institutional review boards (IRBs) within 24 hours
521 of notification in India and 72 hours of notification in Tanzania. Both sites are required to
522 report to WHO within 72 hours of notification. Frequencies and descriptions of SAEs are
523 pooled each month by the data management team at WHO and circulated to investigators,
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524 DSMB and IRBs. When more than half of the person-time is accrued in the study, the DSMB
525 will review an interim data analysis by arm to determine whether stopping boundaries have
526 been crossed.
527 Auditing
528 The sites are responsible for internal quality checks. WHO coordinators conduct at least 1-2
529 site monitoring visits per year to each site.
530 Discussion
531 Zinc supplementation in children during episodes of diarrhea results in several advantages as
532 described earlier [1, 7, 12]. Dose recommendations from WHO/UNICEF were primarily
533 based on previous clinical studies of zinc treatment of diarrhea that used these amounts of
534 zinc. Moreover, because zinc is absorbed less efficiently during diarrhea [17], it has been
535 assumed that the therapeutic dose for treatment of diarrhea should be substantially greater
536 than the RDA for zinc (3-5 mg/d for children <5 years), depending on bioavailability from
537 the usual diet [9]. Therefore, ZTDT was designed to examine the optimal therapeutic dose of
538 zinc supplementation for children under 5 with acute diarrhea in a south Asian country and a
539 sub-Saharan African country.
540 A recent search of clinicaltrials.gov found no other clinical trials that are determining an
541 optimal zinc therapeutic dose for children with acute diarrhea. An additional search on
542 PubMed, Google Scholar and other search engines was carried out where it was noted that
543 one study was found to determine the optimal dose of zinc supplementation in diarrhea [18].
544 The double-blind randomized trial was carried out using 10 or 20 mg zinc sulfate versus
545 placebo. No difference in duration nor frequency of diarrhea was noted. However, this study
546 was carried out in Switzerland, where the epidemiology of acute childhood diarrhea is
547 different than in India or Tanzania. As a result, ZTDT will likely be the first randomized trial
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548 in low resource settings to determine the optimal dose of therapeutic zinc in children with
549 acute diarrhea.
550 Overall, the ZTDT study will measure important clinical outcomes (outcome of diarrhea on
551 growth, frequency of vomiting) in a large number of children under 5 years being treated for
552 acute diarrhea in India and Tanzania. It will also provide evidence for the effect of zinc on
553 plasma zinc concentrations over several days after supplementation, the effect of zinc on
554 post-illness outcomes, as well as the acceptability of zinc supplementation. The trial results
555 will be communicated in academic journals and at the national and local levels in Tanzania
556 and India through policy briefs and dissemination meetings. The results of ZTDT will likely
557 be generalizable to children under 5 with acute diarrhea in similar resource-limited settings
558 and as such will have global relevance. The findings will be an important consideration in
559 any further revision of the WHO technical guidelines on diarrhea case management.
560 Trial status
561 Trial enrollment started on January 16, 2017 and recruitment is ongoing as of December
562 2018.List of abbreviations
563 AAS – Atomic Absorption Spectroscopy
564 AI –Average Intake
565 ANCOVA - Analysis of Covariance
566 CMH – Cochran–Mantel–Haenszel
567 CRFs - Case Report Forms
568 DSMB – Data and Safety Monitoring Board
569 GCP – Good Clinical Practice
570 GEE – Generalized estimating equations
571 GUI - Graphical user interface
572 HIV - Human immunodeficiency virus
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573 IMCI - Integrated Management of Childhood Illness
574 IRBs – Institutional Review Boards
575 ITT – Intention-to-Treat
576 IZINCG – International Zinc Nutrition Consultative Group
577 JMP – Joint Monitoring Programme
578 LMICs – Low- and Middle- Income Countries
579 MUAC – Mid-Upper Arm Circumference
580 ORS – Oral Rehydration Salts
581 QC – Quality Control
582 PP – Per-protocol
583 RDA – Recommended Dietary Allowance
584 RDT – Rapid Diagnostic Test
585 RR – Relative Risk
586 SAE – Serious Adverse Event
587 SEA - South East Asia
588 SQL – Structured Query Language
589 SOP – Standard Operating Procedures
590 SSA – Sub-Saharan Africa
591 UNICEF – United Nations International Children's Emergency Fund
592 WHO – World Health Organization
593 WHZ – Weight for length/height Z-scores
594 ZTDT – Zinc Therapeutic Dose Trial
595596 Ethics approval and consent to participate and publication597598 The ZTDT protocol has been approved by WHO ethics committee (reference no.
599 ERC.0002738, Boston Children’s Hospital IRB (reference no .IRB-P00024269), Tanzania
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600 Food and Drug Authority (reference no. TFDA0016/CTR/0015/03), the Tanzanian National
601 Institute of Medical Research (reference no. NIMR/HQ/R.8a/Vol.IX/2333) and Muhimbili
602 University of Health and Allied Sciences, Dar es Salaam (reference no. 2016-10-
603 31/AEC/Vol.XI/314) and IEC of Subharti Medical College & Hospital, Meerut, UP, India
604 (reference no. SMC/EC/2016/84). All participants will be provided written informed consents
605 before trial enrollment. Caregivers who consent for the trial will also be asked for consent to
606 be escorted back to their home to register the address, to contact the participant, and to be
607 visited at home or followed at home if the participant misses the clinic visit, as well as to
608 store and use all data and child blood samples for this study and future research studies.
609 Availability of data and materials
610 Full protocol is being published in a peer reviewed open access journal. Additional
611 information about the protocol/SOPs will be available on request. In addition, a commitment
612 is made by the investigators: 1) to use data for research purposes and not to identify
613 individual participants and 2) to use appropriate technology to secure the data. Consistent
614 with WHO policies and practice, each site owns the data they generate from the national
615 populations they serve. Sites voluntarily agree to share the data with WHO as the ZTDT
616 Coordinating Body and share the final “frozen data set” with BMGF for their internal use.
617 Access to data
618 WHO and Principal Investigators will have access to final dataset.
619 Declaration of interest
620 The authors declare no competing interests.
621 Funding
622 This study is funded by Bill and Melinda Gates Foundation. The funder has no role in study
623 design, collection, management, analysis and interpretation of data; writing of the report and
624 decision to submit for publication.
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625 Authors’ contributions
626 All the authors participated in conceptualization, design, sample size calculations and writing
627 of the analysis plan for the study. Two workshops held at WHO to develop joint harmonized
628 SOP and CRFs for the study in which all the authors contributed. SSS and PD prepared the
629 first draft of the paper. All authors contributed to revisions of the manuscript and contributed
630 to the revision of the final draft of the manuscript. All the authors have read and approved the
631 final manuscript.
632 Acknowledgements
633 We express our gratitude to the external scientific reviewers of the proposal, for their
634 valuable inputs, which led to important improvements in the study protocol.
635 We appreciate the willingness of the DSMB members Dr. William MacLeod (Chair), Dr.
636 Mario Gheri, Dr. Godwin D. Ndossi; Professor Rosalind Gibson, and Professor Nita Bhandari
637 to provide external study oversight.
638 Additionally authors would like to acknowledge the India core field, data management and
639 lab team: Dr. Pratibha Dixit, Dr. Aishwarya Chauhan, Vishi Saxena, Sanjay Kaushik, Vinod
640 Kumar, Manoj Kumar, Anita Rani and Arvind Sharma and Tanzanian study core team: Dr.
641 Kristina Lugangira, Dr. Abraham Samma, Sr. Juliana Mghamba, Sr. Veneranda Ndesangia,
642 Upendo Kibwana, Cecilia Msemwa, Zachariah Mtulo, Salmin Baleche and all the site staff
643 for their support and hard-work in setting up of field and laboratory systems, piloting testing
644 and SOP development.
645 **Zinc Therapeutic Dose trial (ZTDT) Team (in alphabetical order of sites):
646 India (Center for Public Health Kinetics, New Delhi):
647 Pratibha Dhingra; Usha Dhingra; Arup Dutta; Prabhabati Devi; Jitendra Kumar; Saikat Deb;
648 Om Prakash Semwal and Sunil Sazawal
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649 Tanzania (Muhimbili University of Health and Allied Sciences and Boston Children’s
650 Hospital):
651 Sarah S Somji; Karim Manji; Rodrick Kisenge; Mohamed Bakari; Said Aboud; Enju Liu;
652 Christopher Sudfeld and Christopher Duggan
653 WHO (Maternal Newborn Child and Adolescent Health Department):
654 Per Ashorn; Rajiv Bahl and Jonathon Simon ([email protected])
655 Additional Files:
656 Additional file 1: Figure 1 Consort diagram
657 Additional file 2: Figure 2 Schedule of events
658 Additional file 3: SPIRIT Checklist (Standard Protocol Items: Recommendations for
659 Interventional Trials)
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660 References
661 1. Aggarwal R, Sentz J, Miller MA. Role of zinc administration in prevention of childhood
662 diarrhea and respiratory illnesses: a meta-analysis. Pediatrics. 2007;119(6):1120–30.
663 2. Bhatnagar S, Natchu UCM. Zinc in child health and disease. Indian J Pediatr.
664 2004;71(11):991–5.
665 3. Lukacik M, Thomas RL, Aranda JV. A meta-analysis of the effects of oral zinc in the
666 treatment of acute and persistent diarrhea. Pediatrics. 2008;121(2):326–36.
667 4. Prasad AS. Discovery of human zinc deficiency and studies in an experimental human
668 model. Am J ClinNutr. 1991;53(2):403–12.
669 5. Haider BA, Bhutta ZA. The Effect of Therapeutic Zinc Supplementation among Young
670 Children with Selected Infections: A Review of the Evidence. Food Nutr Bull. 2009
671 ;30(1_suppl1):S41–59.
672 6. Patel A, Mamtani M, Dibley MJ, Badhoniya N, Kulkarni H. Therapeutic Value of Zinc
673 Supplementation in Acute and Persistent Diarrhea: A Systematic Review. PLoS ONE.
674 2010.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860998/. Accessed 21 July 2018.
675 7. Lazzerini M, Wanzira H. Oral zinc for treating diarrhoea in children. Cochrane Database
676 Syst Rev. 2016 20;12:CD005436.
677 8. World Health Organization, Department of Child and Adolescent Health and
678 Development. The treatment of diarrhoea: a manual for physicians and other senior
679 health workers. Geneva: Dept. of Child and Adolescent Health and Development, World
680 Health Organization; 2005.
681 9. Institute of Medicine (US) Panel on Micronutrients. Dietary Reference Intakes for
682 Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese,
683 Molybdenum, Nickel, Silicon, Vanadium, and Zinc [Internet]. Washington (DC):
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684 National Academies Press (US); 2001.
685 http://www.ncbi.nlm.nih.gov/books/NBK222310/Accessed 21 July 2018.
686 10. Strand TA, Chandyo RK, Bahl R, Sharma PR, Adhikari RK, Bhandari N, et al.
687 Effectiveness and efficacy of zinc for the treatment of acute diarrhea in young children.
688 Pediatrics. 2002;109(5):898–903.
689 11. Bahl R, Bhandari N, Saksena M, Strand T, Kumar GT, Bhan MK, et al. Efficacy of zinc-
690 fortified oral rehydration solution in 6- to 35-month-old children with acute diarrhea. J
691 Pediatr. 2002;141(5):677–82.
692 12. Sazawal S, Black RE, Bhan MK, Bhandari N, Sinha A, Jalla S. Zinc supplementation in
693 young children with acute diarrhea in India. NEngl J Med. 1995;333(13):839–44.
694 13. World Health Organization, Department of Maternal N Child and Adolescent Health,
695 World Health Organization. Revised WHO classification and treatment of pneumonia in
696 children at health facilities: evidence summaries. 2014.
697 http://apps.who.int/iris/bitstream/10665/137319/1/9789241507813_eng.pdf?ua=1
698 Accessed 21 July 2018.
699 14. IZiNCG Technical Briefs .IZiNCG. http://www.izincg.org/technical-briefs/Accessed 21
700 July 2018.
701 15. Mauri L, D'Agostino RB, Sr. Challenges in the Design and Interpretation of
702 Noninferiority Trials. N Engl J Med 2017;377(14):1357-67. doi:
703 10.1056/NEJMra1510063.
704 16. WHO/UNICEF Joint Monitoring Programme (JMP) for Water Supply and Sanitation.
705 Improved and unimproved water and sanitation facilities. 2014.
706 http://www.wssinfo.org/definitions-methods/watsan-categories/Accessed 21 July 2018
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707 17. Castillo-Duran C, Heresi G, Fisberg M, Uauy R. Controlled trial of zinc supplementation
708 during recovery from malnutrition: effects on growth and immune function. Am J
709 ClinNutr. 1987;45(3):602–8.
710 18. Crisinel PA, Verga M-E, Kouame KSA, Pittet A, Rey-Bellet CG, Fontaine O, et al.
711 Demonstration of the effectiveness of zinc in diarrhoea of children living in Switzerland.
712 Eur J Pediatr. 2015;174(8):1061–7.
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Assessed for eligibility (n=)
Excluded (n=)
Not meeting inclusion criteria (n=)
Declined to participate (n=) Other reasons (n=)
Randomized (n=)
Allocated to 5 mg Zinc Sulfate (n=)
Allocated to 10 mg Zinc Sulfate (n=)
Allocated to 20 mg Zinc Sulfate (n=)
Withdrawn participation (n=) Discontinued intervention
but follow-up continued (n=) Unable to contact (n=) Died(n=)
Without assessable outcome data (n=)Analyzed (n=)
Without assessable outcome data (n=)Analyzed (n=)
Without assessable outcome data (n=)Analyzed (n=)
Withdrawn participation (n=) Discontinued intervention
but follow-up continued (n=) Unable to contact (n=) Died(n=)
Withdrawn participation (n=) Discontinued intervention
but follow-up continued (n=) Unable to contact (n=) Died(n=)
Enrolment
Allocation
Follow-up
Analysis
Figure 1: Consort Diagram
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STUDY PERIOD
Enrolment Allocation Post-allocation Close-out
TIMEPOINT** 0 0 Day3
Day 5
Day 7
Day 10
Day 15
Day 21
Day 30
Day 45 Day 60
ENROLMENT:
Eligibility screening X
Informed consent X
Randomization X
Allocation X
INTERVENTIONS:
Zinc 5 mg X
Zinc 10 mg X
Zinc 20 mg X
ORS/IV fluids X
ASSESSMENTS:
Baseline demographics X
Infant anthropometrics X X
Breastfeeding status X
Rotavirus vaccination X
Antibiotic use for diarrhea
treatmentX
Zinc Supplement acceptability X
Infant morbidity X X X
Blood plasma levels X X X X X X
Duration of diarrhea X X X X X
Vomiting X X X X X
Fig. 2 Schedule of enrolment, interventions and assessments (SPIRIT Figure).
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SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*
Section/item Item No
Description Addressed on page number
Administrative information
Title 1 Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym Title Page
2a Trial identifier and registry name. If not yet registered, name of intended registry 3Trial registration
2b All items from the World Health Organization Trial Registration Data Set CLINICALTRIALS.GOV IDENTIFIER: NCT03078842
Protocol version 3 Date and version identifier 3
Funding 4 Sources and types of financial, material, and other support 26
5a Names, affiliations, and roles of protocol contributors Title page, 2, 26, & 27 Roles and responsibilities
5b Name and contact information for the trial sponsor DR JONATHON SIMON, WORLD HEALTH ORGANIZATION, 20 AVENUE APPIA, 1211 GENEVA 27 SWITZERLAND; [email protected]
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5c Role of study sponsor and funders, if any, in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication, including whether they will have ultimate authority over any of these activities
26
5d Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint adjudication committee, data management team, and other individuals or groups overseeing the trial, if applicable (see Item 21a for data monitoring committee)
22
Introduction
Background and rationale
6a Description of research question and justification for undertaking the trial, including summary of relevant studies (published and unpublished) examining benefits and harms for each intervention
4, 5
6b Explanation for choice of comparators 5
Objectives 7 Specific objectives or hypotheses 6, 7
Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group), allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory) 6
Methods: Participants, interventions, and outcomes
Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will be collected. Reference to where list of study sites can be obtained
7
Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and individuals who will perform the interventions (eg, surgeons, psychotherapists)
7, 8
11a Interventions for each group with sufficient detail to allow replication, including how and when they will be administered
9, 10Interventions
11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose change in response to harms, participant request, or improving/worsening disease)
11
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11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence (eg, drug tablet return, laboratory tests)
10
11d Relevant concomitant care and interventions that are permitted or prohibited during the trial 10
Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen efficacy and harm outcomes is strongly recommended
17, 18
Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see Figure)
11-15, Figure 2
Sample size 14 Estimated number of participants needed to achieve study objectives and how it was determined, including clinical and statistical assumptions supporting any sample size calculations
8, 9
Recruitment 15 Strategies for achieving adequate participant enrolment to reach target sample size 7
Methods: Assignment of interventions (for controlled trials)
Allocation:
Sequence generation
16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions
11
Allocation concealment mechanism
16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned
11
Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions
11
Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how
11
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17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s allocated intervention during the trial
11
Methods: Data collection, management, and analysis
Data collection methods
18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol
11-16
18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols
14
Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol
16, 17
Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the protocol
18-22
20b Methods for any additional analyses (eg, subgroup and adjusted analyses) 21
20c Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation)
18
Methods: Monitoring
Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed
22
21b Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial
22
Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events and other unintended effects of trial interventions or trial conduct
22
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Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent from investigators and the sponsor
22
Ethics and dissemination
Research ethics approval
24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval 25
Protocol amendments
25 Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes, analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals, regulators)
Not Included
Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and how (see Item 32)
12
26b Additional consent provisions for collection and use of participant data and biological specimens in ancillary studies, if applicable
NA
Confidentiality 27 How personal information about potential and enrolled participants will be collected, shared, and maintained in order to protect confidentiality before, during, and after the trial
12
Declaration of interests
28 Financial and other competing interests for principal investigators for the overall trial and each study site 26
Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that limit such access for investigators
26
Ancillary and post-trial care
30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from trial participation
NA
Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals, the public, and other relevant groups (eg, via publication, reporting in results databases, or other data sharing arrangements), including any publication restrictions
23
31b Authorship eligibility guidelines and any intended use of professional writers Not Included
31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code 26
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Appendices
Informed consent materials
32 Model consent form and other related documentation given to participants and authorised surrogates Available on request
Biological specimens
33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable
NA
*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items. Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons “Attribution-NonCommercial-NoDerivs 3.0 Unported” license.
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Confidential: For Review OnlyEffect of dose reduction of supplemental zinc for childhood diarrhea: study protocol for a double-masked, randomized,
controlled trial in India and Tanzania
Journal: BMJ Paediatrics Open
Manuscript ID bmjpo-2019-000460.R1
Article Type: Protocol
Date Submitted by the Author: 28-Feb-2019
Complete List of Authors: Somji, Sarah; Muhimbili University of Health and Allied Sciences, PediatricsDhingra, Pratibha; Centre for Public Health Kinetics (CPHK)Dhingra, Usha; Centre for Public Health Kinetics (CPHK)Dutta, Arup; Centre for Public Health Kinetics (CPHK)Devi, Prabhabati; Centre for Public Health Kinetics (CPHK)Kumar, Jitendra; Centre for Public Health Kinetics (CPHK)Deb, Saikat; Centre for Public Health Kinetics (CPHK)Semwal, Om Prakash; Centre for Public Health Kinetics (CPHK)Sazawal, Sunil; Centre for Public Health Kinetics (CPHK)Manji, Karim; Muhimbili University of Health and Allied Sciences, PediatricsKisenge, Roderik; Muhimbili University of Health and Allied Sciences, PediatricsBakari, Mohamed; Muhimbili University of Health and Allied Sciences, PediatricsAboud, Said; Muhimbili University of Health and Allied Sciences, PediatricsLiu, Enju; Children's Hospital BostonSudfeld, Chris; Harvard University T H Chan School of Public HealthDuggan, Christopher; Children's Hospital BostonAshorn, Per; Tampereen yliopistoBahl, Rajiv; Organisation mondiale de la SanteSimon, Jonathon; Organisation mondiale de la Sante,
Keywords: General Paediatrics, Paediatric Practice, Pharmacology, Tropical Inf Dis
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1
1 Effect of dose reduction of supplemental zinc for childhood diarrhea: study protocol for
2 a double-masked, randomized, controlled trial in India and Tanzania
3
4 Sarah S Somji* ([email protected])1, Pratibha Dhingra*([email protected])2
5 and the Zinc Therapeutic Dose Trial (ZTDT) Team**
6 * Joint First Authors
7 ** ZTDT Team by alphabetical order of sites:
8 India (Center for Public Health Kinetics, New Delhi):
9 Usha Dhingra ([email protected]); Arup Dutta ([email protected]);
10 Prabhabati Devi ([email protected]); Jitendra Kumar
11 ([email protected]); Saikat Deb ([email protected]); Om Prakash Semwal
12 ([email protected]) and Sunil Sazawal ([email protected])
13 Tanzania (Muhimbili University of Health and Allied Sciences, Dar es Salaam and Boston
14 Children’s Hospital, Boston):
15 Karim Manji ([email protected]); Rodrick Kisenge ([email protected]);Mohamed
16 Bakari ([email protected]); Said Aboud ([email protected]); Enju Liu
17 ([email protected]); Christopher Sudfeld ([email protected]) and
18 Christopher Duggan ([email protected]).
19 WHO (Maternal Newborn Child and Adolescent Health Department):
20 Per Ashorn ([email protected]), Rajiv Bahl ([email protected]) and Jonathon Simon
21 ([email protected]).
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22 Correspondence to: Dr. Jonathon Simon, Department of Maternal, Newborn, Child and
23 Adolescent Health, World Health Organization, 20, avenue Appia, 1211 Geneva 27,
24 Switzerland at [email protected]
25 Affiliations:
26 1. MUHAS-Harvard Projects, Muhimbili University of Health and Allied Sciences,
27 Dar-es-Salaam, Tanzania
28 2. Center for Public Health Kinetics, New Delhi, India
29 3. World Health Organization, Geneva, Switzerland
30 Address for reprints: World Health Organization, 20, avenue Appia, 1211 Geneva 27,
31 Switzerland
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32 Abstract
33 Background: Diarrhea associated mortality and morbidity are highest in infants and young children in low- and
34 middle- income countries (LMICs). Zinc supplementation during acute diarrhea has been shown to reduce the
35 duration of illness and the risk of persistent diarrhea. However, vomiting with zinc supplementation is a common
36 side effect that may interfere with compliance and programmatic scale-up, and may be related to the dose
37 prescribed.
38 Methods/design: The Zinc Therapeutic Dose Trial (ZTDT) is a two-center (Tanzania and India), three-arm
39 randomized, double-blind controlled non-inferiority trial. Children 6-59 months of age with acute diarrhea are
40 eligible to participate. Enrolled children (1500 per arm; 4500 total) will be randomly allocated to receive 5, 10 or
41 20 mg of zinc sulfate daily for 14 days and will be followed up for 60 days after enrollment. All children will
42 receive WHO-UNICEF IMCI standard of care (oral or intravenous rehydration and zinc as indicated and feeding
43 advice). The primary efficacy outcomes of the trial are the percentage of subjects with diarrhea duration more
44 than 5 days, the mean total number of loose or watery stools after enrollment and the proportion of children
45 vomiting within 30 minutes of zinc administration.
46 Discussion: The ZTDT trial will determine the optimal dose of therapeutic zinc supplements for treatment of
47 acute diarrhea in children aged 6-59 months in two LMICs. The results of the trial are likely to be generalizable
48 to childhood acute diarrhea in similar resource-limited settings and may influence global policy about zinc
49 supplementation dosage during acute diarrhea.
50 Trial Registration: ClinicalTrials.gov identifier NCT03078842. Registered on March 13, 2017. Protocol
51 Version/Date: Zinc Protocol Version 4.0/ 19/09/2016.
52 Keywords: Zinc supplementation, Under-five, Children, Optimal dose, Randomized-controlled trial,
53 Acute diarrhea
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WHAT IS ALREADY KNOWN ABOUT THE SUBJECT
Therapeutic zinc supplementation has been recommended by WHO - UNICEF for standard
diarrhea case management since 2004.
Research evidence had shown zinc reduced the duration and severity of the acute diarrhea,
and lowered the risk of persistent diarrhea.
However, many trials noted that current dosage (20 mg/day for children aged >6 months) is
associated with vomiting and possibly poor compliance.
WHAT THIS STUDY HOPES TO ADD:
This study evaluates whether lower doses of zinc (10 mg/5 mg) are non-inferior to the
standard dose in efficacy but superior for vomiting.
If vomiting is less frequent and efficacy is maintained with low dose zinc, caregiver
acceptability may improve, leading to greater utilization and better health outcomes.
54
55 Background:
56 Zinc is a vital micronutrient essential for protein synthesis, cell growth and differentiation,
57 immune function, and intestinal transport of water and electrolytes [1]. Zinc is also important
58 for normal growth and development of children both with and without diarrhea [2]. Zinc
59 deficiency is associated with an increased risk of gastrointestinal infections, altered structure
60 and function of the gastrointestinal tract, and impaired immune function [3-5]. Dietary
61 deficiency of zinc is especially common in low-income countries because of a low dietary
62 intake of zinc-rich foods (including foods of animal origin) or low bioavailability due to the
63 presence of phytates often found in cereal-based diets [1].
64 The benefits of zinc supplementation in the management of diarrhea have been observed in
65 several studies. Patel et al. [6] noted in a systematic review and meta-analysis an approximate
66 20% reduction in the mean duration of acute diarrhea with zinc supplementation. Lazzerini and
67 Wanzira [7], summarizing data from 2581 children from 9 trials, noted that children older than
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68 6 months receiving zinc supplements had a shorter diarrhea duration by about half a day (mean
69 difference -11.46 hours, 95% CI -19.72 to -3.19). A reduction in diarrhea that continues up to
70 day 7 (risk ratio 0.73, 95% CI 0.61 and 0.88) was also noted in this study. Moreover, the study
71 indicated that zinc supplements in children with persistent diarrhea (defined as diarrhea for
72 more than 14 days) also may shorten average diarrhea duration by 16 hours (mean difference -
73 15.84 hours, 95% CI -25.43 to -6.24). As a result of these and other trials, since 2005
74 WHO/UNICEF have recommended routine zinc supplementation (10 mg daily for infants
75 below 6 months of age; 20 mg daily for children older than 6 months) for 10-14 days [8].
76 These doses, however, were not chosen after dose-ranging studies, and substantially exceed the
77 recommended dietary allowances (RDA) for zinc in infancy and early childhood (2-5 mg/day)
78 [9]. High doses of zinc can cause epigastric pain and lethargy, and its metallic taste can also
79 induce vomiting. Vomiting with zinc supplements is a common side effect noted in many
80 published trials. A meta-analysis by Lukacik et al (2008) [3] summarized the findings for acute
81 and persistent diarrhea from a total of 22 zinc intervention trials, using a range of zinc dosing
82 (5 mg to 45 mg/day). In 11 acute diarrhea trials (n= 4438), the proportion of participants who
83 vomited after the initial dose was significantly higher with zinc use than with placebo (12.7%
84 vs. 7.6%; RR: 1.55; 95% CI: 1.30–1.84; P ≤0.001). According to Lazzerini and Wanzira’s
85 review [7], the risk of vomiting increased with zinc supplementation in both children older than
86 age 6 months (risk ratio 1.57, 95% CI 1.32 to 1.86; 2605 children, 6 trials) as well as children
87 younger than 6 months of age (risk ratio 1.54, 95% CI 1.05 to 2.24; 1334 children, 2 trials).
88 Other studies have also reported that therapeutic zinc supplementation during diarrhea causes
89 higher rates of vomiting [10-12].
90 Thus, although substantial evidence supports the efficacy of zinc supplementation during
91 episodes of diarrhea, vomiting is significantly more common among supplemented children.
92 Lower doses of zinc, provided they are equally effective, might have the advantage of reducing
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93 side effects, increasing compliance and programmatic roll-out. If the zinc dose for diarrhea
94 treatment and zinc deficiency can be uniform, then the national essential drug lists can be
95 simplified. Therefore, there exists an important rationale to identify the dose of zinc
96 supplementation associated with both efficacy and an improved safety profile. We present the
97 protocol for a two-center, randomized, double-blind controlled trial of three doses of zinc
98 conducted in Tanzania and India. The overall goals of the study are to evaluate the non-
99 inferiority, safety and acceptability of the two lower zinc doses (5 and 10 mg/day) versus
100 standard zinc dose (20 mg/day).
101 Methods
102 Study design
103 ZTDT is an individually randomized, parallel group, double-blind, controlled trial of three
104 doses of supplemental zinc conducted among 4500 children ages 6-59 months in India and
105 Tanzania (ClinicalTrials.gov identifier NCT03078842).
106 The trial protocol was developed by collaborators at WHO, Department of Maternal Newborn,
107 Child and Adolescent Health (Geneva, Switzerland) together with teams in New Delhi, India
108 (Centre for Public Health Kinetics), Dar es Salaam, Tanzania (Muhimbili University of Health
109 and Allied Sciences) and Boston, USA (Boston Children’s Hospital and Harvard TH Chan
110 School of Public Health). The ZTDT protocol diagram is presented in Additional file 1. The
111 schedule of trial enrollment, interventions, and assessments is presented in Additional file 2.
112 The first participant was enrolled in the trial in January 2017 in India and March 2017 in
113 Tanzania and follow-up data collection will continue through approximately April 2019. This
114 paper follows the Standard Protocol Items: Recommendations for Interventional trials
115 (SPIRIT) checklist (see Additional file 3).
116 The study population will include 4500 children aged 6-59 months with diarrhea of less than
117 72 hours duration. Enrolled children will be followed up for 60 days after randomization. The
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118 participant will be followed up closely for the first 15 days to record post intervention morbidity
119 outcomes and to ensure compliance with study regimen. Study participants will be assessed
120 again at thirty (30), forty-five (45) and sixty (60) days post enrollment to estimate impact on
121 longer-term morbidity outcomes.
122 Patient and Public Involvement
123 This research was done without patient involvement. Patients were not invited to comment on
124 the study design and were not consulted to develop patient relevant outcomes or interpret the
125 results. Patients were not invited to contribute to the writing or editing of this document for
126 readability or accuracy.
127 Primary and secondary objectives
128 The overall aim of ZTDT is to identify the optimal dose of zinc in children under 5 years with
129 acute diarrhea.
130 The primary aims of the study are: 1) to assess whether lower doses of zinc (5 or 10 mg/day)
131 are at least as effective as the standard dose of zinc (20 mg/day) in children 6-59 months, in
132 terms of burden of illness (percentage of subjects with diarrhea lasting more than 5 days; mean
133 number of loose or watery stools during follow-up) and 2) to evaluate the improved
134 effectiveness by reduction in the risk of vomiting in the 30 minutes after receipt of the
135 supplement in low dose supplemental zinc (5 or 10 mg/day) in comparison to standard dose
136 of zinc (20 mg/day).
137 Secondary aims of the study are: 1) to explore whether lower doses of zinc have any impact on
138 post- acute illness morbidity outcomes assessed at 30, 45 and 60 days, 2) to explore whether
139 duration of treatment, estimated by mother’s reports and collection of used zinc blister packs,
140 has any impact on the primary outcomes, 3) to determine the acceptability of low dose
141 supplemental zinc compared to standard zinc dose based on an acceptability scale and 4) to
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142 determine whether the different doses of zinc are associated with differential plasma zinc levels
143 at days 3, 7, 15, 21 and 30 after randomization.
144 Participants/interventions and outcomes
145 Study setting
146 The study recruitment sites are in South East Asia (SEA) and Sub-Saharan Africa (SSA). In
147 SEA the study recruitment sites are located in Sangam Vihar, a resettlement colony on outskirts
148 of South Delhi and Harsh Vihar, a semi-urban locality in North East District, Delhi, India. In
149 SSA recruitment sites are at health centers in Temeke District Hospital, Mbagala Round Table
150 and Mbagala Rangi Tatu, Dar es Salaam, Tanzania. Both the sites will be recruiting from
151 outpatient health facilities.
152 Eligibility
153 Study participants will be eligible for enrollment in the study if they meet the following
154 inclusion criteria: 1) males and female children between 6 to 59 completed months of age, 2)
155 acute diarrhea duration of less than 72 hours at the time of screening (defined as three or more
156 loose or watery stools) or dysentery (defined as visible blood in the stool), 3) likely to stay
157 within the study area for the next 2 months and 4) written informed consent is obtained from
158 caretaker.
159 Children will be excluded if they: 1) have severe acute malnutrition [weight for length/height
160 (WHZ) Z score of <-3 or edema], 2) have severe dehydration that cannot be corrected within
161 4 to 6 hours, 3) have signs of severe pneumonia (characterized by presence of either chest in-
162 drawing and either of the following; convulsions or unconscious or vomiting everything [13],
163 sepsis, rapid diagnostic test (RDT)-confirmed malaria or other severe illness, 4) have
164 previously or currently been enrolled in the study, 5) are currently enrolled in another trial, 6)
165 are not intending to remain in study area for the duration of the study, 7) have a caregiver who
166 declines participation in the study, 8) have other child currently enrolled in the study in the
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167 same household and 9) have used zinc supplements during the three days preceding study
168 enrolment.
169 Sample size
170 We will enroll 4500 children (1500 per study arm) in the trial. Table 1 presents sample size
171 assumptions and calculations for each of the three primary outcomes. The total sample size for
172 the trial was selected to have at least 90% power for each of the primary outcomes. The primary
173 outcomes (mean number of loose or watery stools post-randomization and percentage of
174 children with diarrhea for more than 5 days) are both based on the non-inferiority hypotheses,
175 while the primary outcome of vomiting post-dose assumes a superiority hypothesis. We
176 assumed 1:1:1 randomization between the three treatment arms, a nominal Type I error rate
177 (alpha) of 0.05 and 5% loss to follow-up rate.
178 Table 1 Sample size calculations for the primary trial outcomes
179Outcome Value in
standard dose (20 mg/d) group
Expected value in lower dose groups
Non-inferiority margin
Power, Significance level
Sample size per arm (with ~5% attrition)
Duration of diarrhea, mean (SD)
3.0 (2.7) days 3.0 (2.7) daysLower dose equally effective
0.3 days 90%, 5% 1460
Percentage of children with diarrhea >5 days
16% 16%Lower dose equally effective
+4% 90%, 5% 1500
Total number of loose or watery stools during follow up, mean (SD)
10 (9) stools 10 (9) stoolsLower dose equally effective
+2 stools 90%, 5% 1460
Percentage of children with vomiting during follow up
20% 15%Lower dose safer
Not applicable because of superiority
90%, 5% 1340
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hypothesis for this outcome
180181182 Interventions
183 Children are randomized to receive one of the three zinc sulfate regimens (each taken once
184 daily for 14 days): 1) 5 mg; 2) 10 mg; or 3) 20 mg. The zinc tablet (all 3 regimens) ingredients
185 include aerosil, maize starch, ethylvanillin (a vanilla flavor), aspartame, magnesium stearate
186 vegetal and vivapur. The three study tablets have identical appearance, taste and smell. The
187 supplement is a dispersible tablet that dissolves in water or breast-milk in about 20 seconds.
188 The caretakers are instructed to dissolve the tablet in 5–10 mL of water or breast milk
189 immediately before administration. Children receive zinc supplementation for 14 days. The
190 first dose of study drug is given at the health facility in which the child is enrolled.
191 All three doses of the regimen are packaged identically; 10 tablets in 1 blister, 2 blisters in 1
192 pack (extra doses are provided for redosing in case of vomiting). Each pack is pre-labelled with
193 participant ID. Study regimen is manufactured by Laboratoires Pharmaceutiques Rodael S.A.S,
194 France and shipped to WHO for randomization and labelling, before shipment to recruitment
195 sites (as described below in Randomization, allocation, concealment and masking).
196 Adherence assessment
197 Adherence to the study regimen is optimized by close follow up: either in person (clinic/home
198 visits) or telephone contact on days 3, 5, 7, 10 and 15. Adherence to the regimen is measured
199 by counting the numbers of unused tablets during each follow-up visit. Study workers/ research
200 assistants physically count the tablets during home or clinic visits. We give the
201 mother/caregiver a small booklet to record every successful administration as well as any failed
202 attempts to administer the intervention. Study worker reviews the diary card on each visit and
203 collects filled diary card on day 15 visit.
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204 Standard of care
205 All enrolled children receive standard of care for diarrhea management per WHO/UNICEF,
206 Tanzanian, and Indian Ministry of Health guidelines. This includes standardized assessment of
207 hydration status, rehydration and maintenance fluids with intravenous or oral rehydration
208 solutions (ORS) as indicated, recommendation for increased fluid intake and continued feeding
209 at home, and instructions on when to seek follow-up care. Children with known Human
210 Immunodeficiency Virus (HIV), receive care and treatment appropriate for the child and
211 referral to the Care and Treatment Centre nearest to the child’s home.
212 Randomization, allocation, concealment and masking
213 Trial arm allocation was performed according to a computer-generated randomization list
214 (generated off-site by a non-study statistician) of 4500 individuals, stratified by country and
215 child age (>6 - <24 months versus >24 - <60 months). A statistician will hold the randomization
216 list codes until completion of primary analyses or until requested by the Data and Safety
217 Monitoring Board (DSMB). Within each stratum, subjects are randomly assigned equally in
218 permuted blocks of variable size to receive one of the three doses of zinc. The purpose of the
219 stratification is to have equal distribution of younger and older children among all three
220 treatment groups. An independent pharmacy team prepared the zinc regimen in Geneva by
221 labeling blister packs with participant IDs based on the randomization list. Labels were printed
222 at WHO, Geneva, and enrolled children were assigned sequential serial numbers. These pre-
223 labeled regimens were shipped to the sites in Tanzania and India. The packing and physical
224 appearance of the three study regimes are identical. In addition, the three doses of the regimen
225 are similar in taste, appearance and smell. As a result, the research teams, study staff as well as
226 caregivers are fully blinded to treatment arm. Zinc formulations are serially numbered
227 according to the randomization codes. The code is kept in a password protected spreadsheet
228 file by WHO personnel and is not available to the investigators until the end of the study. This
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229 randomization list was converted into unique sequential serial numbers for each enrolled child
230 at each site. This code will only be broken if the DSMB requests this information for a
231 participant [should they suspect a relationship of a suspected Severe Adverse Event (SAE) to
232 the study drug] or for a study arm (in their interim analyses).
233 Data collection, management and analysis
234 Screening and randomization
235 Both sites will be recruiting from community clinics. Surveillance systems are set up at clinics
236 to identify all children presenting with diarrhea. Trained study staff stationed at each health
237 facility are responsible for identification of children who are potentially eligible. The study
238 team is trained in good clinical practice (GCP) as well as confidentiality and research ethics.
239 Written informed consent is sought from the caretaker of children meeting the eligibility
240 criteria. A child can only be enrolled once into the study. The informed consent process
241 includes telling the caretaker that they are free to withdraw from the study at any time, and that
242 participation is entirely voluntary.
243 Following consent, each participant will be assigned a unique subject identification number. A
244 card detailing the subject ID and the health personnel responsible for enrollment will be
245 provided to the caregiver of enrolled children. The patient locator form /subject master log will
246 be the only link between the subject ID and the participant’s name. This information will be
247 stored together with the consent in a locked file cabinet and an online database accessible to
248 only the Project Manager and required study staff for the purpose of patient tracking.
249 A baseline assessment, including a detailed physical examination (record of vital signs, axillary
250 temperature, respiratory rate, pulse/rate, hydration and nutritional status), will be performed by
251 a research physician at the time of screening for enrollment. The child is evaluated for any
252 Integrated Management of Childhood Illness (IMCI) general danger signs (unable/ able to
253 drink or breastfeed, convulsions, vomiting, unconscious, stiff neck) and evidence of
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254 dehydration or pneumonia, as well as information on recent medical history (duration of
255 diarrhea, bloody stool/dysentery, recent fever). Vital signs (temperature/heart rate/respiratory
256 rate) in dehydrated child are recorded after the child is rehydrated and stabilized and remaining
257 clinical assessment/ screening is completed thereafter. Anthropometry measurements will be
258 taken after rehydration and stabilization as required, before completing the screening for
259 enrolling the participant. Weight will be measured using an electronic scale with a sensitivity
260 of ±10 g by two independent observers; length (for children less than 24 months old) or height
261 (for children 24 months or older) which will be measured using a length board to within 0.1
262 cm. Mid-upper arm circumference (MUAC) will be measured using non-stretchable tape to the
263 nearest 0.1 cm. All anthropometric measures will be recorded in duplicate (India) and triplicate
264 (Tanzania). Once these measurements and other eligibility criteria are met, the subject will get
265 enrolled.
266 Baseline socio-demographic information and vaccination history
267 Parents of enrolled subjects will be interviewed at study clinic or at home for the baseline socio-
268 demographic characteristics viz. parent's literacy level and occupation, type of house,
269 possession of assets and possible exposures (recent antibiotic use, travel history, water use and
270 filtration and sanitation), breastfeeding and vaccination history and information is recorded in
271 the case record forms.
272 Follow-up visits
273 Scheduled follow-up visits begin from the third day of the enrolment and are generated daily
274 via an automated visit generation module of the data management software. All children
275 enrolled at the study clinic are to be followed up in the clinic or at home by a trained field
276 worker on day 3, 5, 7, 10, 15, 30, 45, and 60 (Additional file 1 and 2). In India, study staff will
277 visit the household of enrolled children for all follow up visitations. If the child is not available
278 at home, a phone call will be made to follow-up the child. In Tanzania, a participant will be
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279 followed up at home or clinic on Days 3, 7, 15, 30 and 60 and by phone on days 5, 10 and 45.
280 Children still having diarrhea after Day 15 are treated according to standard management
281 guidelines [8]. At each visitation during active follow up period, a study worker asks the mother
282 or other caregiver about the following and records the responses in a predesigned form/data
283 entry module:
284 1. Ongoing symptoms of diarrheal illness, including the number and consistency of stools
285 2. Vomiting episodes including any episode within 30 minutes following zinc administration.
286 3. Adherence with zinc study regimen ascertained via mothers’ recall and/or reported use of
287 zinc tablets by the child in the Diarrhea Diary Card and cross-checked by determining the
288 number of unused tablets.
289 4. Any adverse events, including hospital admissions and deaths of study participants.
290 5. Criteria for returning to clinic (e.g. lethargy, poor oral intake, dehydration).
291 6. Any other inter-current illness
292 7. Reported use of antibiotics and IV/other fluids
293 At day 30, 45, and 60 post-enrolment, the study participants are assessed either at the study
294 clinic, via telephone or with a home visit for presence of any illness, diarrhea, fever, cough, or
295 fast or difficult breathing within last 24 hours and any time in past 2 weeks. On the Day 60
296 study completion clinic visit, in addition to morbidity assessment, anthropometric
297 measurements will be taken for each participant using the same procedure as mentioned in the
298 baseline assessment.
299 Children not available on the visitation day will be visited the next day; if they are still not
300 available, they are followed-up until found or deemed lost to follow-up. Caregivers are asked
301 to contact the study physicians if they feel that their children are sick between the visits.
302 Children sick on the day of visitation will be referred to the study clinic for a detailed morbidity
303 assessment as well as treatment by the study physician. During each visit a record of all the
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304 medicines and IV fluids given, information on number and consistency of stools and data on
305 adherence is recorded in the predesigned forms. Close monitoring will be accomplished
306 through repeated follow-ups during the 14-day regimen period. Every effort will be made to
307 track and successfully follow-up all randomized children.
308 Study withdrawal or discontinuation
309 The participants have the right to withdraw from the study at any time. The subjects will be
310 considered withdrawn from the trial in case of death or lost to follow-up. However, every effort
311 will be made to track and successfully follow-up all enrolled children. The information on
312 withdrawn or discontinued participants will be recorded through a withdrawal slip by field
313 worker and clinic supervisor.
314 Blood sampling and laboratory investigations
315 To allow the measurement of the potential differential impact of the varying zinc doses on
316 plasma zinc levels during the follow-up period, each study participant will provide two blood
317 samples. One third of the study participants will have blood draws at Days 1 and 15, one third
318 at Days 3 and 21, and the remaining one third on Days 7 and 30 post-enrolment for assessment
319 of plasma zinc levels.
320 Blood drawing will follow the protocols of the International Zinc Nutrition Consultative Group
321 (IZINCG). These include use of powder-free gloves, zinc-free syringes and butterfly, needles
322 and trace element free blood tubes [14]. Plasma is separated at 15 minutes after blood collection
323 and aliquots are transferred into trace element-free Eppendorf plastic tubes for temporary
324 storage at -20°C and then shipped to central lab for final storage at -80°C in India. In Tanzania,
325 plasma samples are stored in site refrigerators at 2-8oC and then shipped to central laboratory
326 storage at -80oC by the end of the day. Plasma zinc concentrations will be measured by atomic
327 absorption spectroscopy (AAS-400 Perkin Elmer, USA).
328 Quality assurance
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329 The persons with at least a secondary school degree will be recruited for data collection team.
330 The following quality assurance checks will be done regularly to ensure highest level of data
331 integrity and quality:
332 ● An extensive 1-3 weeks training session for data collectors to ensure the inter-data collector
333 reliability is above 95%.
334 ● Pilot field data testing is monitored closely by the research scientist(s) responsible for
335 quality assurance.
336 ● Real time electronic data capture will ensure data validation such as range, logical checks
337 and data integrity.
338 ● Interim aggregate data analysis will be carried out periodically for data pattern, heaping,
339 missing values, etc. Based on the feedback, effective measures will be taken to
340 eliminate/minimize the errors.
341 For overall visit supervision, quality control (QC) supervisors will perform quality checks by
342 visiting at least 10% of the households, re-interviewing 5% respondents and cross-checking
343 this data with concerned field worker. QC supervisors are primarily responsible for overall
344 field related project activities (data quality and integration at all levels, resolving community
345 issues), and training. Computer randomized list of enrolled children will be used by quality
346 control team for random household visits.
347 The QC team works in close coordination with the investigative team and provides regular
348 feedback on quality issues. Quarterly meetings between quality control team and senior
349 investigative team are scheduled to discuss overall quality issues and related corrective
350 measures to be undertaken.
351 Data management
352 Data are collected in real-time using either tablets or laptop computers. Each site uses an in-
353 house designed data management software with user friendly graphical user interface (GUI) for
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354 data collection. Generic case report forms (CRFs) and data dictionary are used to maintain data
355 consistency across sites. In-built range, logical and skipping pattern will ensure data quality and
356 integrity.
357 The database is password protected with restricted access. Each user is assigned a unique user
358 identifier to access the software and an audit trial is maintained for database operations. The
359 centralized server located at each site synchronizes the data on daily basis. Data are shared
360 monthly with WHO data quality monitoring team who is responsible for data quality checks
361 and provide feedback to sites. They generate query forms which are returned to the study sites
362 for verification.
363 Data security and storage
364 A backup of the data is kept in the server at the WHO protected by a specific password and
365 accessible to only authorized users. An additional backup of the data is kept in a password
366 protected external hard drive at a site away from the WHO at the recruiting site central offices.
367 Record retention and archival
368 All the study documents including participant’s source data and documents will be archived by
369 the study sites after the completion of the study, till the time WHO informs in writing to the
370 study sites that they no longer need to maintain the study documents.
371 Outcome measures
372 The primary efficacy outcomes are 1) the percentage of enrolled children who have duration
373 of diarrhea of more than 5 days, 2) the mean number of loose or watery stools after enrolment
374 and 3) the percentage of children vomiting within 30 minutes of administration of the zinc
375 tablet.
376 Secondary outcomes of the trial include: i) proportion of children experiencing serious adverse
377 events (life-threatening or hospitalization; ii) proportion with intermediate duration of diarrhea
378 (diarrhea continuing beyond three days)); iii) Proportion of guardians with positive attitude
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379 towards treatment; iv) treatment adherence; v) mean serum plasma zinc concentration at days
380 1, 3, 7, 15, 21 and 30; vi) illness symptoms between day 15 and 60 after the treatment 2-week
381 period prevalence and number of days with diarrhea, fever or respiratory symptoms. vii) 60-
382 day change in height/length, weight, MUAC, height/length-for-age z-score, weight-for-
383 length/height z-score, weight-for-age z-score, MUAC z-score, stunting, wasting, underweight;
384 viii) adherence to regimen; ix) study dropouts; x) maternal report of ease in supplement
385 administration; xi) maternal report of child acceptability; xii) maternal recommendation of use
386 for other children; xiii) maternal report of change in child skin condition, child appetite, activity
387 levels, mood, and diarrhea severity.
388 Statistical analysis plan
389 All primary analyses of non-inferiority tests will be conducted on the principle of intention-to-
390 treat (ITT). All non-inferiority margins will be defined a priori. We will also conduct
391 sensitivity analyses using a per-protocol (PP) approach [15]. The analysis population for the
392 ITT approach will include all randomized patients with assessable data, regardless of whether
393 the participant is known to have received a full 14-day dose of zinc or not. The analysis
394 population for the PP approach will include study participants who are documented to have
395 taken zinc supplementation for the first 5 days after randomization regardless if they vomited
396 the dose. The PP definition was selected due to the primary outcome being diarrhea duration
397 >5 days. Descriptive statistics will be used to summarize background demographic and
398 baseline clinical characteristics by trial arm.
399 Analysis of Primary Outcomes
400 Primary Outcome 1: Number (%) of subjects with diarrhea episodes >5 days. The last day of
401 diarrhea will be defined as the day in which there is passage of the last liquid or semi-liquid
402 stool prior to two diarrhea-free days (days when less than 3 loose stools / day). The duration of
403 diarrhea will be defined as the number of days between randomization (not the reported day of
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404 onset of diarrhea symptoms before randomization) and the last day with diarrhea. Children who
405 are lost to follow-up, die, or withdraw before 5 days will be excluded from this analysis. For
406 the primary analysis, we have set the threshold for non-inferiority of lower doses of zinc (10
407 mg/d or 5 mg/d) as compared to the standard 20 mg/d at less than 4% in absolute risk (risk
408 difference) compared to the standard dose in the proportion of subjects with prolonged
409 diarrhea. The proportions of participants with a prolonged diarrhea episode (defined as >5
410 days) will be calculated in all treatment arms, and the risk differences (and their 95% CI’s)
411 between the lower doses (5 mg/d and 10 mg/d) and the standard dose (20 mg) will be estimated.
412 A Cochran–Mantel–Haenszel (CMH) approach will be used to take stratifying randomization
413 factors, e.g. country and age groups, into account. If the upper limit of the 95% CI for the risk
414 difference is less than 4%, we will conclude non-inferiority for one or both lower dose arms.
415 We will also examine the relative risk (RR) of prolonged diarrhea episodes in both lower dose
416 arms compared to the standard dose arm using relative risk regression which accounts for
417 factors included in the stratified randomization. In secondary analyses, Cox proportional hazard
418 models will be used to estimate hazard ratios and their 95% confidence intervals for recovery
419 from diarrhea in each of the lower dose arms compared to the standard dose arm. The time to
420 recovery is defined as the duration of diarrhea as noted above.
421 Primary Outcome 2: Total number of loose or watery stools after enrolment. An Analysis of
422 Covariance (ANCOVA) approach will be used to estimate the mean differences and their 95%
423 CI in the total number of loose or watery stools after enrollment for each lower dose arm
424 compared to the standard dose arm, while accounting for the stratified randomization design.
425 The non-inferiority margin was set at 2 stools, therefore if the upper limit of the 95% CI is <2
426 stools, we will reject the null hypothesis that the lower dose zinc is inferior to the standard dose
427 in term of reducing the total number of loose or watery stools.
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428 Primary Outcome 3: Vomiting within 30 minutes after administration of the trial regimen. We
429 hypothesize that the lower dose of zinc will be associated with a reduced risk of vomiting and
430 therefore a superiority test will be used. Vomiting within 30 minutes of each daily zinc dose is
431 repeatedly measured during the 14-day administration period. Generalized estimating
432 equations (GEE) with the log link, binomial variance, and exchangeable correlation matrix will
433 be used to assess the relative risk of vomiting among the three treatment arms. The
434 exchangeable working covariance matrix and robust estimators of the variances will be used to
435 construct confidence intervals. The robust estimators are consistent estimators of the variances
436 even if the working correlation matrix is mis-specified. Stratified randomization factors will be
437 considered in the model. A secondary analysis concerning severe vomiting (defined as
438 vomiting requiring hospitalization or vomiting requiring intravenous fluids) will also be
439 performed in similar fashions. We will also examine the proportion of children ever vomiting
440 between the treatment arms and examine whether the relative risk of vomiting differs by
441 follow-up day.
442 Analysis of change in plasma zinc and other secondary outcomes
443 Due to the sampling design of participants having paired blood sampled on three schedules
444 (days 1 and 15; days 3 and 21; days 7 and 30), we will assess the effect of the randomized zinc
445 regimen at both the population- and individual-level. For the analysis of population-level zinc,
446 we will use a mixed model approach to compare patterns of plasma zinc concentrations over
447 time between the randomized treatment arms. The main exposure variables in the model will
448 include treatment arm, study day, and an interaction term between treatment arm and study
449 day. In case there is not a linear relationship between study day and plasma zinc concentrations,
450 restricted cubic splines will be used to examine the non-linear relationship between these two
451 variables. The interaction term between zinc dose and study day in the model measures whether
452 the trajectory of plasma zinc differs by zinc supplementation over the full period. If the overall
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453 test for this interaction term is statistically significant, group means at each assessment day will
454 then be compared using least-square means with the Tukey-Kramer adjustment. To be
455 consistent with our stratified randomization study design, stratification factors will be included
456 in the model.
457 For all other secondary outcomes (see clinicaltrial.gov NCT03078842), we will use approaches
458 similar to the primary analyses, including an ANCOVA approach for non-repeated continuous
459 outcomes, relative risk regression for non-repeated binomial outcomes, and GEE or mixed
460 effect models for repeated longitudinal outcomes.
461 Sensitivity analyses and effect modification of treatment effect
462 Although randomization is designed such that, on average, treatment groups will be balanced
463 with respect to all baseline prognostic factors, this may not be true in all cases. As a result, we
464 will re-assess treatment effects as observed in the original analysis after adjusting for factors
465 that are potentially imbalanced across randomized arms. If imbalance occurs, we will compare
466 and present the results for both unadjusted and adjusted analyses.
467 In addition, the study was not designed to detect effect modification of the treatment effect
468 among subgroups of children. As a result, we acknowledge that we are likely underpowered to
469 detect differences in the magnitude of a treatment effect among subgroups unless there is strong
470 effect modification. The potential baseline effect modifiers we plan to explore include: study
471 site (India/Tanzania), child age (<12 months vs. >12 months), child sex (male vs. female), prior
472 receipt of rotavirus vaccine (Yes vs. No), maternal HIV status (Yes vs. No), dysentery at
473 enrollment (Yes vs. No), baseline dehydration status (Yes vs. No), baseline axillary
474 temperature (<38°C vs. >38°C), baseline respiratory rate, recent antibiotic use (Yes vs. No),
475 inter-current illness (cough, or difficulty breathing, or earache), current breastfeeding status
476 (Yes vs. No), and prior breastfeeding status (Yes vs. No). Additional variables that we will test
477 for effect modification will include child anthropometric measures (length/height for age,
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478 weight for age, and weight for length/height Z scores, and MUAC), family socioeconomic
479 status (<median wealth index, >median wealth index), maternal education, household water
480 quality (improved vs. unimproved), household sanitation quality (improved vs. unimproved),
481 baseline plasma zinc levels (<median, > median), and trial regimen adherence (<median,
482 >median). Factor analysis will be used to derive wealth index from family possession variables.
483 UNICEF/WHO JMP definitions will be used to clarify improved vs. unimproved for the
484 household water quality and sanitation [16].
485 Monitoring
486 Data and Safety Monitoring Board (DSMB)
487 A Data Safety and Monitoring Board has been established to monitor severe adverse events
488 (SAEs) and to review the statistical analysis plan and associated stopping rules for benefit,
489 futility, or harm determined using O’Brien-Fleming stopping boundaries. The DSMB includes
490 5 members with expertise in clinical trials, statistics, child mortality assessment, pediatric care
491 in resource limited settings, or practical experience from each study country. The DSMB meets
492 electronically or in person at least every 6 months. Severe adverse events (SAEs) related to
493 study participation are monitored in real-time, are summarized and reported to study
494 investigators, WHO and relevant institutional review boards (IRBs) within 24 hours of
495 notification in India and 72 hours of notification in Tanzania. Both sites are required to report
496 to WHO within 72 hours of notification. Frequencies and descriptions of SAEs are pooled each
497 month by the data management team at WHO and circulated to investigators, DSMB and IRBs.
498 When more than half of the person-time is accrued in the study, the DSMB will review an
499 interim data analysis by arm to determine whether stopping boundaries have been crossed.
500 Auditing
501 The sites are responsible for internal quality checks. WHO coordinators conduct at least 1-2
502 site monitoring visits per year to each site.
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503 Discussion
504 Zinc supplementation in children during episodes of diarrhea results in several advantages as
505 described earlier [1, 7, 12]. Dose recommendations from WHO/UNICEF were primarily based
506 on previous clinical studies of zinc treatment of diarrhea that used these amounts of zinc.
507 Moreover, because zinc is absorbed less efficiently during diarrhea [17], it has been assumed
508 that the therapeutic dose for treatment of diarrhea should be substantially greater than the RDA
509 for zinc (3-5 mg/d for children <5 years), depending on bioavailability from the usual diet [9].
510 Therefore, ZTDT was designed to examine the optimal therapeutic dose of zinc
511 supplementation for children under 5 with acute diarrhea in a south Asian country and a sub-
512 Saharan African country.
513 A recent search of clinicaltrials.gov found no other clinical trials that are determining an
514 optimal zinc therapeutic dose for children with acute diarrhea. An additional search on
515 PubMed, Google Scholar and other search engines was carried out where it was noted that one
516 study was found to determine the optimal dose of zinc supplementation in diarrhea [18]. The
517 double-blind randomized trial was carried out using 10 or 20 mg zinc sulfate versus placebo.
518 No difference in duration nor frequency of diarrhea was noted. However, this study was carried
519 out in Switzerland, where the epidemiology of acute childhood diarrhea is different than in
520 India or Tanzania. As a result, ZTDT will likely be the first randomized trial in low resource
521 settings to determine the optimal dose of therapeutic zinc in children with acute diarrhea.
522 Overall, the ZTDT study will measure important clinical outcomes (outcome of diarrhea on
523 growth, frequency of vomiting) in a large number of children under 5 years being treated for
524 acute diarrhea in India and Tanzania. It will also provide evidence for the effect of zinc on
525 plasma zinc concentrations over several days after supplementation, the effect of zinc on post-
526 illness outcomes, as well as the acceptability of zinc supplementation. The trial results will be
527 communicated in academic journals and at the national and local levels in Tanzania and India
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528 through policy briefs and dissemination meetings. The results of ZTDT will likely be
529 generalizable to children under 5 with acute diarrhea in similar resource-limited settings and
530 as such will have global relevance. The findings will be an important consideration in any
531 further revision of the WHO technical guidelines on diarrhea case management.
532 Trial status
533 Enrollment begin in January 2017 and follow-up is estimated to be completed in April 2019.
534 As of February 1, 2019, 742 children are still contributing data to the ZTDT study.
535 List of abbreviations
536 AAS – Atomic Absorption Spectroscopy
537 AI –Average Intake
538 ANCOVA - Analysis of Covariance
539 CMH – Cochran–Mantel–Haenszel
540 CRFs - Case Report Forms
541 DSMB – Data and Safety Monitoring Board
542 GCP – Good Clinical Practice
543 GEE – Generalized estimating equations
544 GUI - Graphical user interface
545 HIV - Human immunodeficiency virus
546 IMCI - Integrated Management of Childhood Illness
547 IRBs – Institutional Review Boards
548 ITT – Intention-to-Treat
549 IZINCG – International Zinc Nutrition Consultative Group
550 JMP – Joint Monitoring Programme
551 LMICs – Low- and Middle- Income Countries
552 MUAC – Mid-Upper Arm Circumference
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553 ORS – Oral Rehydration Salts
554 QC – Quality Control
555 PP – Per-protocol
556 RDA – Recommended Dietary Allowance
557 RDT – Rapid Diagnostic Test
558 RR – Relative Risk
559 SAE – Serious Adverse Event
560 SEA - South East Asia
561 SQL – Structured Query Language
562 SOP – Standard Operating Procedures
563 SSA – Sub-Saharan Africa
564 UNICEF – United Nations International Children's Emergency Fund
565 WHO – World Health Organization
566 WHZ – Weight for length/height Z-scores
567 ZTDT – Zinc Therapeutic Dose Trial
568569 Ethics approval and consent to participate and publication570571 The ZTDT protocol has been approved by WHO ethics committee (reference no.
572 ERC.0002738, Boston Children’s Hospital IRB (reference no .IRB-P00024269), Tanzania
573 Food and Drug Authority (reference no. TFDA0016/CTR/0015/03), the Tanzanian National
574 Institute of Medical Research (reference no. NIMR/HQ/R.8a/Vol.IX/2333) and Muhimbili
575 University of Health and Allied Sciences, Dar es Salaam (reference no. 2016-10-
576 31/AEC/Vol.XI/314) and IEC of Subharti Medical College & Hospital, Meerut, UP, India
577 (reference no. SMC/EC/2016/84) (see Additional file 4). All participants will be provided
578 written informed consents before trial enrollment. Caregivers who consent for the trial will also
579 be asked for consent to be escorted back to their home to register the address, to contact the
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580 participant, and to be visited at home or followed at home if the participant misses the clinic
581 visit, as well as to store and use all data and child blood samples for this study and future
582 research studies.
583 Availability of data and materials
584 Full protocol is being published in a peer reviewed open access journal. Additional
585 information about the protocol/SOPs will be available on request. In addition, a commitment
586 is made by the investigators: 1) to use data for research purposes and not to identify
587 individual participants and 2) to use appropriate technology to secure the data. Consistent
588 with WHO policies and practice, each site owns the data they generate from the national
589 populations they serve. Sites voluntarily agree to share the data with WHO as the ZTDT
590 Coordinating Body and share the final “frozen data set” with BMGF for their internal use.
591 Access to data
592 WHO and Principal Investigators will have access to final dataset.
593 Declaration of interest
594 The authors declare no competing interests.
595 Funding
596 This study is funded by Bill and Melinda Gates Foundation (see Additional file 5). The
597 funder has no role in study design, collection, management, analysis and interpretation of
598 data; writing of the report and decision to submit for publication.
599 Authors’ contributions
600 All the authors participated in conceptualization, design, sample size calculations and writing
601 of the analysis plan for the study. Two workshops held at WHO to develop joint harmonized
602 SOP and CRFs for the study in which all the authors contributed. SSS and PD prepared the
603 first draft of the paper. All authors contributed to revisions of the manuscript and contributed
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604 to the revision of the final draft of the manuscript. All the authors have read and approved the
605 final manuscript.
606 Acknowledgements
607 We express our gratitude to the external scientific reviewers of the proposal, for their valuable
608 inputs, which led to important improvements in the study protocol.
609 We appreciate the willingness of the DSMB members Dr. William MacLeod (Chair), Dr. Mario
610 Gheri, Dr. Godwin D. Ndossi; Professor Rosalind Gibson, and Professor Nita Bhandari to
611 provide external study oversight.
612 Additionally authors would like to acknowledge the India core field, data management and
613 lab team: Dr. Pratibha Dixit, Dr. Aishwarya Chauhan, Vishi Saxena, Sanjay Kaushik, Vinod
614 Kumar, Manoj Kumar, Anita Rani and Arvind Sharma and Tanzanian study core team: Dr.
615 Kristina Lugangira, Dr. Abraham Samma, Sr. Juliana Mghamba, Sr. Veneranda Ndesangia,
616 Upendo Kibwana, Cecilia Msemwa, Zachariah Mtulo, Salmin Baleche and all the site staff
617 for their support and hard-work in setting up of field and laboratory systems, piloting testing
618 and SOP development.
619 **Zinc Therapeutic Dose trial (ZTDT) Team (in alphabetical order of sites):
620 India (Center for Public Health Kinetics, New Delhi):
621 Pratibha Dhingra; Usha Dhingra; Arup Dutta; Prabhabati Devi; Jitendra Kumar; Saikat Deb;
622 Om Prakash Semwal and Sunil Sazawal
623 Tanzania (Muhimbili University of Health and Allied Sciences and Boston Children’s
624 Hospital):
625 Sarah S Somji; Karim Manji; Rodrick Kisenge; Mohamed Bakari; Said Aboud; Enju Liu;
626 Christopher Sudfeld and Christopher Duggan
627 WHO (Maternal Newborn Child and Adolescent Health Department):
628 Per Ashorn; Rajiv Bahl and Jonathon Simon ([email protected])
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629 Additional Files:
630 Additional file 1: Figure 1 Consort diagram
631 Additional file 2: Figure 2 Schedule of events
632 Additional file 3: SPIRIT Checklist (Standard Protocol Items: Recommendations for
633 Interventional Trials)
634 Additional file 4: Letters of ethical approval
635 Additional file 5: Letter from the funder
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636 References
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641 3. Lukacik M, Thomas RL, Aranda JV. A meta-analysis of the effects of oral zinc in the
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643 4. Prasad AS. Discovery of human zinc deficiency and studies in an experimental human
644 model. Am J ClinNutr. 1991;53(2):403–12.
645 5. Haider BA, Bhutta ZA. The Effect of Therapeutic Zinc Supplementation among Young
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657 9. Institute of Medicine (US) Panel on Micronutrients. Dietary Reference Intakes for Vitamin
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660 Academies Press (US); 2001. http://www.ncbi.nlm.nih.gov/books/NBK222310/Accessed
661 21 July 2018.
662 10. Strand TA, Chandyo RK, Bahl R, Sharma PR, Adhikari RK, Bhandari N, et al.
663 Effectiveness and efficacy of zinc for the treatment of acute diarrhea in young children.
664 Pediatrics. 2002;109(5):898–903.
665 11. Bahl R, Bhandari N, Saksena M, Strand T, Kumar GT, Bhan MK, et al. Efficacy of zinc-
666 fortified oral rehydration solution in 6- to 35-month-old children with acute diarrhea. J
667 Pediatr. 2002;141(5):677–82.
668 12. Sazawal S, Black RE, Bhan MK, Bhandari N, Sinha A, Jalla S. Zinc supplementation in
669 young children with acute diarrhea in India. NEngl J Med. 1995;333(13):839–44.
670 13. World Health Organization, Department of Maternal N Child and Adolescent Health,
671 World Health Organization. Revised WHO classification and treatment of pneumonia in
672 children at health facilities: evidence summaries. 2014.
673 http://apps.who.int/iris/bitstream/10665/137319/1/9789241507813_eng.pdf?ua=1
674 Accessed 21 July 2018.
675 14. IZiNCG Technical Briefs .IZiNCG. http://www.izincg.org/technical-briefs/Accessed 21
676 July 2018.
677 15. Mauri L, D'Agostino RB, Sr. Challenges in the Design and Interpretation of Noninferiority
678 Trials. N Engl J Med 2017;377(14):1357-67. doi: 10.1056/NEJMra1510063.
679 16. WHO/UNICEF Joint Monitoring Programme (JMP) for Water Supply and Sanitation.
680 Improved and unimproved water and sanitation facilities.
681 2014. http://www.wssinfo.org/definitions-methods/watsan-categories/Accessed 21 July
682 2018
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683 17. Castillo-Duran C, Heresi G, Fisberg M, Uauy R. Controlled trial of zinc supplementation
684 during recovery from malnutrition: effects on growth and immune function. Am J
685 ClinNutr. 1987;45(3):602–8.
686 18. Crisinel PA, Verga M-E, Kouame KSA, Pittet A, Rey-Bellet CG, Fontaine O, et al.
687 Demonstration of the effectiveness of zinc in diarrhoea of children living in Switzerland.
688 Eur J Pediatr. 2015;174(8):1061–7.
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Assessed for eligibility (n=)
Excluded (n=)
• Not meeting inclusion criteria
(n=)
• Declined to participate (n=)
• Other reasons (n=)
Randomized (n=)
Allocated to 5 mg
Zinc Sulfate (n=)
Allocated to 10 mg Zinc
Sulfate (n=)
Allocated to 20 mg Zinc
Sulfate (n=)
• Withdrawn participation (n=)
• Discontinued intervention
but follow-up continued (n=)
• Unable to contact (n=)
• Died(n=)
Without assessable outcome
data (n=)
Analyzed (n=)
Without assessable outcome
data (n=)
Analyzed (n=)
Without assessable outcome
data (n=)
Analyzed (n=)
• Withdrawn participation (n=)
• Discontinued intervention
but follow-up continued (n=)
• Unable to contact (n=)
• Died(n=)
• Withdrawn participation (n=)
• Discontinued intervention
but follow-up continued (n=)
• Unable to contact (n=)
• Died(n=)
Enrolment
Allocation
Follow-up
Analysis
Figure 1: Consort Diagram
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STUDY PERIOD
Enrolment Allocation Post-allocation Close-out
TIMEPOINT** 0 0 Day
3
Day
5
Day
7
Day
10
Day
15
Day
21
Day
30
Day
45 Day 60
ENROLMENT:
Eligibility
screening X
Informed consent X
Randomization X
Allocation X
INTERVENTIONS:
Zinc 5 mg X
Zinc 10 mg X
Zinc 20 mg X
ORS/IV fluids X
ASSESSMENTS:
Baseline
demographics X
Infant
anthropometrics X
X
Breastfeeding
status X
Rotavirus
vaccination X
Antibiotic use for
diarrhea
treatment
X
Zinc Supplement
acceptability X
Infant morbidity X X X
Blood plasma
levels X X X X X X
Duration of
diarrhea X X X X X
Vomiting X X X X X
Fig. 2 Schedule of enrollment, interventions and assessments (SPIRIT Figure).
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SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents*
Section/item Item No
Description Addressed on page number
Administrative information
Title 1 Descriptive title identifying the study design, population, interventions, and, if applicable, trial acronym Title Page
Trial registration 2a Trial identifier and registry name. If not yet registered, name of intended registry 3
2b All items from the World Health Organization Trial Registration Data Set CLINICALTRIALS.GOV
IDENTIFIER:
NCT03078842
Protocol version 3 Date and version identifier 3
Funding 4 Sources and types of financial, material, and other support 26
Roles and
responsibilities
5a Names, affiliations, and roles of protocol contributors Title page, 2, 26, & 27
5b Name and contact information for the trial sponsor DR JONATHON
SIMON, WORLD
HEALTH
ORGANIZATION, 20
AVENUE APPIA, 1211
GENEVA 27
SWITZERLAND;
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5c Role of study sponsor and funders, if any, in study design; collection, management, analysis, and
interpretation of data; writing of the report; and the decision to submit the report for publication, including
whether they will have ultimate authority over any of these activities
26
5d Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint
adjudication committee, data management team, and other individuals or groups overseeing the trial, if
applicable (see Item 21a for data monitoring committee)
22
Introduction
Background and
rationale
6a Description of research question and justification for undertaking the trial, including summary of relevant
studies (published and unpublished) examining benefits and harms for each intervention
4, 5
6b Explanation for choice of comparators 5
Objectives 7 Specific objectives or hypotheses 6, 7
Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group),
allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory)
6
Methods: Participants, interventions, and outcomes
Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will
be collected. Reference to where list of study sites can be obtained
7
Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and
individuals who will perform the interventions (eg, surgeons, psychotherapists)
7, 8
Interventions 11a Interventions for each group with sufficient detail to allow replication, including how and when they will be
administered
9, 10
11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose
change in response to harms, participant request, or improving/worsening disease)
11
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11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence
(eg, drug tablet return, laboratory tests)
10
11d Relevant concomitant care and interventions that are permitted or prohibited during the trial 10
Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood
pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation
(eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen
efficacy and harm outcomes is strongly recommended
17, 18
Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits
for participants. A schematic diagram is highly recommended (see Figure)
11-15, Figure 2
Sample size 14 Estimated number of participants needed to achieve study objectives and how it was determined,
including clinical and statistical assumptions supporting any sample size calculations
8, 9
Recruitment 15 Strategies for achieving adequate participant enrolment to reach target sample size 7
Methods: Assignment of interventions (for controlled trials)
Allocation:
Sequence
generation
16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any
factors for stratification. To reduce predictability of a random sequence, details of any planned restriction
(eg, blocking) should be provided in a separate document that is unavailable to those who enrol
participants or assign interventions
11
Allocation
concealment
mechanism
16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered,
opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned
11
Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to
interventions
11
Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome
assessors, data analysts), and how
11
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17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant’s
allocated intervention during the trial
11
Methods: Data collection, management, and analysis
Data collection
methods
18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related
processes to promote data quality (eg, duplicate measurements, training of assessors) and a description
of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known.
Reference to where data collection forms can be found, if not in the protocol
11-16
18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be
collected for participants who discontinue or deviate from intervention protocols
14
Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality
(eg, double data entry; range checks for data values). Reference to where details of data management
procedures can be found, if not in the protocol
16, 17
Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of
the statistical analysis plan can be found, if not in the protocol
18-22
20b Methods for any additional analyses (eg, subgroup and adjusted analyses) 21
20c Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any
statistical methods to handle missing data (eg, multiple imputation)
18
Methods: Monitoring
Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement
of whether it is independent from the sponsor and competing interests; and reference to where further
details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is
not needed
22
21b Description of any interim analyses and stopping guidelines, including who will have access to these
interim results and make the final decision to terminate the trial
22
Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse
events and other unintended effects of trial interventions or trial conduct
22
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Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent
from investigators and the sponsor
22
Ethics and dissemination
Research ethics
approval
24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval 25
Protocol
amendments
25 Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes,
analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals,
regulators)
Not Included
Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and
how (see Item 32)
12
26b Additional consent provisions for collection and use of participant data and biological specimens in
ancillary studies, if applicable
NA
Confidentiality 27 How personal information about potential and enrolled participants will be collected, shared, and
maintained in order to protect confidentiality before, during, and after the trial
12
Declaration of
interests
28 Financial and other competing interests for principal investigators for the overall trial and each study site 26
Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that
limit such access for investigators
26
Ancillary and post-
trial care
30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from
trial participation
NA
Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals,
the public, and other relevant groups (eg, via publication, reporting in results databases, or other data
sharing arrangements), including any publication restrictions
23
31b Authorship eligibility guidelines and any intended use of professional writers Not Included
31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code 26
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Appendices
Informed consent
materials
32 Model consent form and other related documentation given to participants and authorised surrogates Available on request
Biological
specimens
33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular
analysis in the current trial and for future use in ancillary studies, if applicable
NA
*It is strongly recommended that this checklist be read in conjunction with the SPIRIT 2013 Explanation & Elaboration for important clarification on the items.
Amendments to the protocol should be tracked and dated. The SPIRIT checklist is copyrighted by the SPIRIT Group under the Creative Commons
“Attribution-NonCommercial-NoDerivs 3.0 Unported” license.
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