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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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Confidential: For Review Only

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

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):


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


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

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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.

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687 Effectiveness and efficacy of zinc for the treatment of acute diarrhea in young children.

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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

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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=)



Withdrawn participation (n=) Discontinued intervention

but follow-up continued (n=) Unable to contact (n=) Died(n=)

Without assessable outcome data (n=)Analyzed (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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2

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


BMJ Paediatrics Open on M

arch 15, 2020 by guest. Protected by copyright.

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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:


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]).


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):


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


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

637 1. Aggarwal R, Sentz J, Miller MA. Role of zinc administration in prevention of childhood

638 diarrhea and respiratory illnesses: a meta-analysis. Pediatrics. 2007;119(6):1120–30.

639 2. Bhatnagar S, Natchu UCM. Zinc in child health and disease. Indian J Pediatr.

640 2004;71(11):991–5.

641 3. Lukacik M, Thomas RL, Aranda JV. A meta-analysis of the effects of oral zinc in the

642 treatment of acute and persistent diarrhea. Pediatrics. 2008;121(2):326–36.

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

646 Children with Selected Infections: A Review of the Evidence. Food Nutr Bull. 2009

647 ;30(1_suppl1):S41–59.

648 6. Patel A, Mamtani M, Dibley MJ, Badhoniya N, Kulkarni H. Therapeutic Value of Zinc

649 Supplementation in Acute and Persistent Diarrhea: A Systematic Review. PLoS ONE.

650 2010.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860998/. Accessed 21 July 2018.

651 7. Lazzerini M, Wanzira H. Oral zinc for treating diarrhoea in children. Cochrane Database

652 Syst Rev. 2016 20;12:CD005436.

653 8. World Health Organization, Department of Child and Adolescent Health and

654 Development. The treatment of diarrhoea: a manual for physicians and other senior health

655 workers. Geneva: Dept. of Child and Adolescent Health and Development, World Health

656 Organization; 2005.

657 9. Institute of Medicine (US) Panel on Micronutrients. Dietary Reference Intakes for Vitamin

658 A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese,

659 Molybdenum, Nickel, Silicon, Vanadium, and Zinc [Internet]. Washington (DC): National

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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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31

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=)


data (n=)

Analyzed (n=)


data (n=)

Analyzed (n=)





• Died(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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1

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;

[email protected]

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2

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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3

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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4

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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5

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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6

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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