Efficiency and Safety of Zinc Sulphate to Reduce the Duration of Acute Diarrheal Disease Between 6 and 59 Months of Age
Phase: NA
Conditions: Diarrhoea;Acute, Diarrhea, Infantile
Sponsor: Instituto Nacional de Salud Publica, Mexico

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Efficacy and safety of zinc sulfate to reduce the duration of 
acute diarrheic disease between 6 and 59 months of age  
 
Project number 1474 
 
Responsible Researcher 
Dr. Jorge Slameron 
52 777 100 13 64 
jorge.salmec@gmail.com 
Dr. Edgar Sanchez Uribe 
Edgar.su@gmail.com 
Dr. Mario Efrain Flores Aldana 
Mario.flores@insp.mx 
Dr. Marcelino Esparza Aguilar 
Marea.b1@gmail.com  
 
Center of Investigation in Population Health 
 

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INDEX 
Abstract ........................................................................................................................... 3 
Backgrounds .................................................................................................................... 4 
Epidemiology and impact of acute diarrheic disease ................................................. 4 
Zinc use in ADD ........................................................................................................... 4 
Zinc physiology ............................................................................................................ 5 
Safety .......................................................................................................................... 5 
Efficacy of zinc usage .................................................................................................. 6 
Zinc effect in acute diarrheic disease ......................................................................... 7 
Zinc as a public health strategy ................................................................................... 8 
Problem Statement ......................................................................................................... 9 
Justification ................................................................................................................... 10 
Research questions ....................................................................................................... 10 
Objectives...................................................................................................................... 10 
General objective .......................................................................................................... 10 
Primary Objectives .................................................................................................... 10 
Secondary Objectives ................................................................................................ 11 
Hypothesis................................................................................................................. 11 
Methods .................................................................................................................... 11 
General study design ................................................................................................ 11 
Study population ....................................................................................................... 12 
Sampling .................................................................................................................... 12 
Analysis Plan.............................................................................................................. 23 
Flowchart .................................................................................................................. 27 
Confidentiality ........................................................................................................... 28 
Risk ............................................................................................................................ 28 
Feasibility ...................................................................................................................... 30 
Bibliography .................................................................................................................. 31 
 
 
 

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Abstract 
Acute diarrheal diseases (ADD) continue to be a public health problem. Even though the oral 
rehydration salts (ORS) have reduced annual death rates related to this, from 4.5 to 1.8 million, 
in the world, it still is the second cause of death in children under the age of 5 years, and in 
Mexico the fifth, generating in the country 20.8% of all the appointments and 10% of all 
pediatric hospitalizations.  
Worldwide in 2004 zinc deficiency was associated to 400,000 deaths in children under the age of 
5, whilst in Mexico, in the ENSANUT-2006, 11.4% of children under the age of 5 had a low zinc 
ingest and 29.2% of children under the age of 2 years had zinc deficiency.  
In Mexico the administration of ORS and immunization against rotavirus has positively impacted 
ADD mortality in children, nonetheless it continues to be a public health issue, being between 
the main causes of morbidity and mortality, with an important load for the health systems. 
Consequently there exists a necessity for the implementation of alternate strategies for this 
condition, nutrition being on of the pillars to strengthen.  
Zinc administration during the ADD episode is a simple intervention for clinical picture reduction 
and relapse. The recommendation of including zinc in the treatment of ADD is not part of the 
Mexican normativity. The evaluation of the efficacy of zinc administration in Mexican children is 
insufficient and a program or national strategy for zinc treatment does not exist.  
Objective. Evaluate the efficacy of oral zinc sulfate administration (20 mg/day for 10 days), 
during the ADD clinic picture in children 6 to 59 months of age treated in nine Sentinel Centers  
of Mexico City.  
Methodology. Randomized clinical trial, multicenter, double risk, controlled. Including 516 
children 6 to 59 months of age, in two groups, 258 in the zinc intervened group, and 258 in the 
controlled group.  
For the analysis descriptive statistic will be used. The comparison of evaluation time of ADD and 
number of evacuations will be done by the use of the Student t test for independent groups in 
case the distribution is normal, or with the Manny-Whitney U test if the distribution is not 
normal. For the evaluation of risk of relapse relative risk will be estimated, reduction of relative 
risk and reduction of absolute risk. The efficacy of the intervention over the duration of the 
disease and the number of evacuations will be model with the lineal general model to adjust by 
the basal characteristics.  
The present proposal will allow to respond to the necessity of counting with controlled studies 
in Mexican children to know the impact of zinc in the reduction of ADD severity, duration and 
relapse.  
 
 
 
 
 
 
 

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Backgrounds 
Epidemiology and impact of acute diarrheic disease  
Acute diarrheal diseases (ADD) are still an important public health problem; worldwide it is the 
second cause of death in children under the age of 5, preceded only by low respiratory 
infections. In Mexico it occupies fifth place amongst the causes of mortality in children under 
the age of five years and generates 20.8% of consultations in health services and 10% of 
pediatric hospitalizations in the country.1  
The disease´s rates are very similar amongst the different states of the Mexican Republic. 
According to ENSANUT 2012, the proportion of ADD in rural areas was 12.3%; and in urban 
areas 10.5% respectively. When the proportion of children under the age of five years of age 
was analyzed by socio economic level, that presented diarrhea in the two weeks previous to the 
interview, it was reported a 10.5%, 11.9%, 11.4%, 10.9% and 10.1% in the socio economic levels 
1, 2, 3, 4, 5, respectively, once again there are no differences between the different socio 
economic levels.2  
Oral rehydration electrolytes and oral rehydration therapy, adopted by the WHO and UNICEF at 
the end of the 70s, have been key in mortality rate descend amongst children under the age of 5 
with acute diarrhea because it went from 4.5 million to 1.8 million annual deaths.3 Nonetheless, 
despite this notable descend, acute diarrhea still is one of the main infant mortality causes in 
developing countries. 4  
It is estimated that 21% of world population is at risk of zinc deficiency according to the food 
supply balance reports.5 
The World Health Organization has recognized zinc deficiency as a risk factor for children´s 
health, since it is associated with diarrhea morbidity in a 10%, low respiratory infections in a 6%, 
and with malaria in 18%, plus 0.8 million deaths in children every year.6  
 
Zinc use in ADD 
Clinic evidence shows that, worldwide, frequent zinc consumption helps prevent ADD.7 The new 
recommendations for the treatment of diarrhea, published by the Global Program of the WHO 
in Genève, are aimed to reduce morality in children under 5 years of age.  This new 
recommendations have in mind two recent transcendental advances: the demonstration of the 
higher efficacy of the new formulation of the ORS with low concentrations of glucose and salt; 
and the good results of the administration od zinc supplements.7 8 9 
The National Nutrition Survey of 1999 reported a prevalence of low levels of zinc of 34% in 
Mexican children under the age of 2 years. 10 Furthermore, in 2006 the National Nutrition Survey 
(ENSANUT) 11 found that the 11.4% of children under the age of 5 years had a zinc intake lower 
than the Estimated Required Average and 29.2% of the children under the age of 2 years 
presented zinc deficiency.12 Zinc deficiency was associated with approximately 400,000 deaths in 
children under the age of 5 years old in 2004 worldwide.13  
 
 

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Zinc physiology 
Moderated zinc deficiency in children of preschool age is common in developing countries; 12 14 it 
may delay growth, inhibit immunological response, suppress appetite, and reduce the capacity 
to detect flavors.15 16 
Zinc has a crucial function in the metalloenzymes, the polyribosomes, the membrane and 
cellular function, which performs an important function in cellular growth and in the function of 
the immune system. Zinc use for the treatment of acute diarrhea affects immune functioning, 
the intestinal structure or its function, and the process of epithelial recuperation during 
diarrhea. Moreover, it has been highlighted that the losses of intestinal zinc during diarrhea 
aggravate the lack of preexisting zinc. 17 
 
Safety 
In the extensive safety studies conducted on laboratory animals, it has been demonstrated that 
zinc is not carcinogenic, mutagenic, or teratogenic.18  
The human body has efficient homeostatic mechanisms that regulate the absorption and 
retention of zinc, which diminishes the probability of toxic accumulation in the body.19  
The toxicity of zinc in adults may occur following a moderately high zinc ingest (> 150mg / day, 
approximately 10 times the daily recommendation) during a long period of time or by ingest of 
more than 1 g of zinc, or by overdose via supplementation or intravenous feeding.20  
High zinc dosage during long periods of time may conduct to a minor concentration of plasmatic 
lipoproteins and diminish the absorption of copper. The descend of copper status may also 
inhibit transportation of iron and give place to anemia.21  
In the most extreme cases of ingest of more than 1 gram of zinc taken daily during many 
months, most of the patients recovered from all symptoms and signs, including fatigue, 
gastrointestinal discomfort and anemia as soon as the ingest of zinc was reduced, and serum 
zinc went back into the normal range.  
The recommended dosage of 10mg in children under the age of 6 months and 20mg in children 
from 6 months to 59 months of age for 10- 14 days have demonstrated effectiveness and safe 
for treatment during diarrhea. Up to this day there has not been reports of serious adverse 
reactions of any kind of zinc supplements for the treatment of diarrhea. The trials have included 
more than 8.500 children that have participated in efficacy trails. The zinc dosages varied from 
5- 45 mg a day, and have been well tolerated in diverse environments. The trails have not found 
differences in the adverse reactions based on the different zinc salts used in the supplement 
trails, which are acetate sulfate and zinc gluconate.  
For the moment, the only secondary effect of the supplement administration has been vomit. 
Out of the seven trails that have informed about vomiting incidence, only two reported more 
vomiting in children with zinc supplements vs. placebo.22 23  
One trail found a significant tendency to diminish copper status when comparing children 
supplemented with zinc and children supplemented without zinc.27Nonetheless, this children 
were malnourished and with persistent diarrhea at the beginning of the study. In general, there 

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is no evidence that short-term zinc supplements as a treatment for diarrhea affect negatively 
copper concentrations.  
Other studies where zinc efficacy has been evaluated for the treatment of pneumonia, malaria, 
measles, and common cold, have not reported serious adverse events related to zinc 
supplement.  
In studies with long-term supplementation in children seeking to improve growth, and prevent 
pneumonia, diarrhea and malaria, it was used 5 to 20 mg Zn/day for a year; there were no 
reports of adverse events in those studies.28 
Like with any treatment, medication or vitamin, zinc supplements should be kept at a safe 
location to prevent accidental ingestion of more of the recommended dosage. In the rare event 
that a child consumes multiple zinc supplements, it is probable that he or she will rapidly vomit. 
There is no evidence that suggest that more adverse events will occur, but like with any 
accidental medication ingestion, the child should be taken to a medical attention provider. The 
risk of more adverse events that are produced from the recommended supplementation 
schemes is extremely low, but the zinc supplement should be maintained at a safe location to 
prevent unnecessary accidents.  
Zinc supplement is a safe and effective treatment fro diarrhea. Zinc also has demonstrated being 
safe in long-term supplementation studies. The most serious adverse events from the 
supplementation trails have been vomiting in some cases and a mild decrease in copper status 
in some children.  None of them have demonstrated to cause long-term damage. Even though in 
adults there has been cases reported of excessive zinc consumption, even the adverse events in 
those cases have been limited to short-term morbidity and few have resulted in long-term 
sequels.  
 
Efficacy of zinc usage  
In the last two decades there has been multiple studies done focused on zinc use in diarrhea; 
they have concluded the following: 
 
Children treated with zinc have a significantly faster recovery than children that do not 
received it (the duration of diarrhea is reduced approximately a 20-25%). On the other 
hand, the risk of severe episodes of more than seven days of duration is reduced in a 
20%. The decrease of the total amount of evacuations goes from 18% to 59% in children 
treated with zinc when compared with the placebo group.29 30 
 
The administration of zinc supplements to children in developing countries, being in a 
continuous way or in short duration treatments, is associated to substantial reductions 
of diarrhea rates.8 
 
Children with persistent diarrhea that took zinc supplements had a probability of 
continuing with diarrhea lower than 24% (Confidence Interval of 95% [CI]: 9% to 37%), 
and the failure rate of the treatment or death was 42% lower (CI of 95%: 10% to 63%) 
than the witness group.8  
 
The studies conducted in children with acute shigellosis have revealed that the 
administration of supplements of zinc favors significantly seroconversion, meaning the 

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appearance of antibodies against shigella, and increases the proportion of circulating B 
lymphocytes and plasmatic cells, as well as the specific response due to the A 
immunoglobulin production (IgA). 31 Consequently it is clear that zinc supplements must 
be administered as a complement to treatment with antibiotic for bloody diarrhea.  
It has been concluded that the administration of zinc supplements has an important and 
beneficial effect in clinic evolution of acute diarrhea, because it reduces severity as well as its 
duration. The administration of zinc supplements for the treatment of diarrhea, together with 
educational programs may reduce inadequate use of antibiotics that are leading to the 
appearance of pathogenic agents resistant to antimicrobials.32 
It has been concluded that the administration of zinc supplements, in a daily dosage from 10 to 
20 mg during 10 to 14 days, is effective in terms of reducing significantly the severity of diarrhea 
and the duration of the episode.  It has not been determined yet the optimal dosage, 
nonetheless it does not appear to be an increase in efficacy when passing from the usual daily 
dosage of 20 to a higher of 30 to 40. It has been demonstrated that a daily dosage of 20 mg of 
elemental zinc is innocuous and efficient, even for lactating infants, and consequently it is 
recommended.  
It has been accomplished the same efficacy with the different zinc salts used (sulfate, acetate 
and zinc gluconate). 33 However, zinc sulfate is low cost, efficient and safe, and consequently 
could be optimal for a national program.34 
 
Zinc effect in acute diarrheic disease 
Zinc deficiency in immune response is likely to increase susceptibility of children with infectious 
diarrhea. There have been two populations phenotypically different of T lymphocytes identified 
that are zinc sensible: the response Th1, important in the protection against intracellular 
infections, and the Th2 response is important in the protection against non invasive infections 
such as helminthes. An adequate ingest of zinc and energy leads to a Th1 dominant response, 
and to a Th2 response regulated to the low. 
In-vitro studies and studies in patients with zinc deficiency have demonstrated that zinc 
performs an essential part mediated by cells and humoral immunity. The findings during zinc 
deficiency are a decrease in lymphocytes numbers (lymphopenia), lymphocytic development 
deterioration, reduced proliferation, apoptosis increase and thymic atrophy.  Zinc is an essential 
cofactor for timulin that induces various T cells markers and promotes the function of T cells, 
including allogenic cytotoxicity, suppressor functions and the production of Il-2. Also modulates 
the liberation of cytokines by the nuclear cells of peripheral blood and induces the proliferation 
of T CD8 cells, that function ass cytotoxic cells capable of recognizing and killing pathogens. 
Zinc also plays a key part in the maintenance of cells from the intestinal mucous. Zinc blocks 
basolateral potassium channels (K+) and therefore inhibits the secretion of liquid dependent of 
the chloride induced by cAMP, an important control point for liquid loss in the large intestine. 
The mechanisms by which zinc may act as an enteroprotector have not been determined yet. 
Zinc also inhibits ion secretion induced by enterotoxin induced the cholera toxin, but not the 
thermo stable enterotoxin induced by E.cola. The cAMP acts as the intracellular effector from 

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thermo labile enterotoxin inducing fluid secretion. So zinc efficacy will be limited to the diarrhea 
induced by thermo labile enterotoxin or to diarrhea mediated by cAMP, but not by cGMP or by 
intracellular calcium. It has also been reported that ZnT-1, zinc transporter, modulates cation 
permeability through the channel of L type calcium (LTCC), therefore the regulation of cationic 
homeostasis. Therefore, ZnT-1 may perform a part in cell ion homeostasis conferring protection 
against pathophysiological events related to cellular permeability of calcium or of zinc. A 
micromolar concentration of extracellular zinc might trigger a massive liberation of calcium from 
the intracellular reserves in colonocytes. A sustained increase of intracellular calcium level can 
also increase the flow of K and a hyperpolarization of the cellular membrane potential, giving 
place to an electrical gradient advantageous for chloride secretion. 
Protozoan intestinal infections by Giardia and Cryptosporidium are common in humans all over 
the world. Especially important are infections in children, during pregnancy, and amongst 
people with HIV/AIDS. The associated morbidity and mortality are high, with more than 58 
million cases of infant protozoan diarrhea every year. A recent study performed in children 
habitants of Mexico City informed that vitamin A and zinc reduced the incidence of G.lamblia, 
while zinc supplements only decrease diarrhea associated with Entamoeba histolytica. 
 
Even though bacterial pathogens may cause diarrheic illnesses, a group of less than 10 (including 
Shigella spp, ETEC, Vibrio cholerae and possibly Camplylobacter jejuni) represent a significant 
percentage of these illnesses in developing countries.  
Crane et al. informed that zinc decreased the adherence of enteropathogenic bacterias of E.coli 
(EPEC) the rabbit intestinal epithelium.35 Also demonstrated that zinc inhibited a key enzyme, 
the ecto-5´-neuclotidase, implicated in the conversion of 5´-AMP in adenosine at the intestine 
lumen. Adenosine triggers fluid secretion of the intestinal cells of the host and also has growth 
promoting effects in the EPEC bacteria. The inhibition of zinc reduced the secretory response 
that triggers watery diarrhea activated by EPEC.  
In animal testing, the net secretion of water and sodium induced by the cholera toxin was four 
times higher in zinc deficiency in comparison with animals suitable for zinc.  
Zinc supplementation reduced significantly the duration of acute shigellosis, promotes a better 
weight increase during recovery and reduces diarrheic morbidity during the following 6 months.  
 
Zinc as a public health strategy 
To obtain maximum impact over diarrheic diseases, zinc and the ORS must be placed within the 
reach of all the community. In the market there are many vitamin preparations and other 
nutritional supplements that contain zinc. Nevertheless, they rarely contain the recommended 
dosage. Consequently, is required a product that contains only zinc, even though copper may be 
added. The product should ne elaborated in a manner that masks the metallic flavor that zinc 
can leave for children to accept it. Zinc salts in tablet form or syrup are the most convenient for 
infant administration.  
Currently in Mexico in the Basic table and the Medication Catalogue 2014 diverse presentations 
exist (medical food, formulas, multivitamins, human milk substitutes and/or diets) that contain 

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diverse concentrations of Zinc Sulfate, fundamental Zinc, Zinc Chloride and/or Zinc Phosphate in 
combination with other active substances. There is only one presentation of multivitamins 
(polyvitamins) and Oral Solution Minerals for children of 6 to 24 months of age where every 100 
ml contains among others vitamins and minerals; monohydrated zinc sulphate (2.744 g) 
equivalent to 1.0 g of fundamental zinc and its use is indicated in patients with specific 
deficiencies and/or pathologies related to deficiency, however in the treatment of Acute 
Diarrheic Disease it is necessary an oral solution as supplement that is zinc based (such as 
heptahydrate sulfate).34 All the supplements distributed to “Prospera” beneficiaries 
(“Nutrisano”, “Vitaniño” and “Nutrivida”) already include a recommended amount of zinc for 
this population. The recommendation to include zinc in ADD treatment is not yet a part of the 
Mexican normativity in the matter. However, there exist background in the health sector that 
have carried out this practice, it has been recommended in the Attention to Nutrition Integral 
Strategy (EsIAN) from the CNPSS through the Promoters Manual and in the Manual for Acute 
Diarrheic Diseases, Prevention, Control, and Treatment of the Health Ministry. Therefore it is 
necessary to look for the way to include zinc in combination with “Vida Suero Oral” to perform a 
more efficient treatment for diarrhea amongst children under the age of five.  
The WHO recommends giving zinc to any malnourished child. UNICEF and the WHO 
recommends zinc supplementation, 10mg, in children under 6 months of age and 20mg in 
lactating children older than 6 months old and preschoolers for 10 to 14 days, as a universal 
treatment for children with diarrhea.  
In Mexico 78% of children population under the age of 5 years live in urban areas. Additionally, 
50% of all the cases of ADD in 2015 in children under the age of 5 years were reported in the 
following states: Distrito Federal, Mexico, Guanajuato, Guerrero, Oaxaca, Jalisco, Puebla. 
A key step to evaluate the effect of zinc supplementation is to conduct a clinical study to 
measure the effect on ADD in children in Mexico. Therefore, the ideal scenery for this evaluation 
would be in an infant population that represents the majority of Mexican population, 
fundamentally constituted by urban or conurbated area inhabitants, where a high population 
density of the targeted group exists and counts with higher case reports.  
 
Problem Statement 
Acute diarrheic disease in our country occupies the fifth place amongst mortality causes in 
children under the age of 5 and generates 20.8% of consultations in health services and 10% of 
pediatric hospitalization.  
Oral rehydration therapy, adopted by the WHO and UNICEF have been key in the decrease of 
the mortality rate amongst children under the age of 5 year with acute diarrhea, despite this 
decrease, acute diarrhea still is one of the main causes of infant mortality in developing 
countries. The administration of zinc supplements has an important and beneficial effect in the 
clinical evolution of acute diarrhea, because it reduces so much its severity as its duration.  
The strategy has been efficient in malnourished children, but the universalization of this strategy 
is still controversial in places where there is a low prevalence of malnutrition.  

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In our country the ADDs are still a public health problem, being amongst the main causes of 
morbidity and mortality, with an important load to the health systems, so there is the need of 
implementation of alternate strategies for this condition, nutrition being one of the pillars to 
strengthen.  
 
Justification  
In Mexico there have been strategies placed, such as the administration of ORS and vaccination 
against rotavirus, which have impacted positively in ADD mortality amongst children. Even 
though the magnitude of the problem has decreased, acute diarrheic disease continues to be 
one of the first causes of infant morbidity and mortality, generating an important load to the 
health systems, being a latent public health problem; therefore there exist the necessity of 
implementation of alternate strategies to combat the incidence of this condition, being nutrition 
one of the pillars to strengthen.  
Zinc administration during an ADD episode is a relatively simple intervention that may allow the 
reduction of severity, duration, and relapse. The anti infectious potential of zinc may be of high 
importance in the actual financial crisis scene of the health systems, and the new and re-
emerging infectious diseases.  
The Integral Strategy of Nutrition Attention (EsIAN) of the CNPSS through the Manual for 
Promoters and in the Manual for the Acute Diarrheic Diseases, Prevention, Control and 
Treatment from the Health Ministry consider zinc supplementations for ADD treatment. 
Nevertheless, to this day, studies that evaluate the efficacy of zinc administration in Mexican 
children are scarce and do not count with any national program or strategy of zinc treatment.  
We consider that the present proposal will allow us to respond to the need of counting with 
controlled studies in Mexican children to know the impact of zinc use in the reduction of ADD 
severity, duration and relapse. 
 
Research questions   
Zinc supplement administration (20 mg/day) in acute diarrheic disease decreases the severity 
and duration of the clinic picture and relapse in children from 6 to 59 months of age, in relation 
with children from the same age group treated with placebo? 
 
Objectives  
General objective   
Evaluate the efficacy of oral zinc sulfate administration (20 mg/day for 10 days), during the ADD 
clinic picture in children from 6 to 59 months of age attended in nine Sentinel Centers of Mexico 
City.  
Primary Objectives 
a) Determine zinc serum concentrations in children from 6 to 59 months of age.  

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b) Compare the effect of 20 mg of zinc oral administration in ADD in the duration of 
diarrhea, in relation with the group that received placebo.  
c) Compare the effect of 20 mg of zinc oral administration in acute diarrheic disease in the 
number of evacuations during the acute diarrheic disease picture, in relation with the 
placebo group. 
 Secondary Objectives  
a) Compare the effect of 20 mg zinc oral administration in acute diarrheic disease in the 
reduction of diarrheic disease recurrence in the subsequent months to the treated 
clinical picture  (from 3 to 6 months), in relation with the placebo group.  
b) Correlate the increase in the zinc serum concentration to the oral treatment for 10 days 
with 20mg of zinc with the duration of ADD picture, the number of total evacuations 
and relapse rates.  
 Hypothesis 
o The oral administration of 20 mg of zinc will decrease at least 12 hours in average the 
duration time of ADD duration in children from 6 to 59 months of age in comparison 
with the placebo group.  
o The oral administration of 20 mg of zinc will decrease at least 30% of diarrheic 
evacuations in children from 6 to 59 months of age in comparison with the placebo 
group. 
Methods 
General study design  
Randomized clinical study, multicenter, double risk, controlled.  
a) Participants: There will be a study done in 516 children, both sexes between 6 to 59 
months of age, that are attended in the sentinel centers assigned for the study (258 for 
each length of the treatment).  
b) Intervention duration: 10 days.  
c) Inclusion period: 6 months (2017). 
d) Average tracking duration to evaluate relapse: 6 months average.  
e) Location: 9 Health Units from the Federal and State Ministries. 
  
Table 1. Sentinel Centers  
Medical unit 
Instituto Nacional de Pediatria 
Hospital Pediátrico de Tacubaya 
Hospital Pediatrico de Iztapalapa 
Centro de Salud Topilejo 
Hospital Pediatrico de Coyoacan 
Hospital Materno Infantil de Xochimilco 

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Table 1. Sentinel Centers  
Medical unit 
Hospital Pediatrico La Villa 
Centro de Salud Cultura Maya 
Centro de Salud San Andres Totoltepec 
Study population 
Children of both sexes, that have between 6 to 59 months of age and that attend a consultation 
for ADD.  
Eligible population  
Children of both sexes, that are between 6 to 59 months of age and that attend a consultation 
for ADD in the Sentinel Health Units (see Table 1) between June and December 2017.  
 
Selection Criteria 
Inclusion Criteria 
Children, both sexes, from 6 to 59 years of age that attend a consultation in the sentinel centers, 
with a Acute Diarrheic Disease diagnose, that both parents or legal guardian agree to participate 
in the study and sign the informed consent letter. That parents have not planned moving to 
other location in a time period of at least a year.  
Exclusion Criteria  
Children with deficient intestinal absorption Syndrome, Acrodermatitis Enterohepatic, Zinc 
Sulfate Hypersensitivity, Leucine metabolic Disorders, secondary lactose intolerance, 
Galactosemia lactase primary deficiency, allergy to cow milk protein, and/or children that are 
supplemented with zinc for the last 6 months.  
 Elimination Criteria 
Children that present hypersensitivity to zinc supplement, diagnosed subsequently to the intake 
with deficient intestinal absorption Syndrome, Acrodermatitis Enterotropic, Zin Sulfate 
hypersensitivity, leucine metabolism disorders, secondary lactose intolerance, Galactosemia 
lactase primary deficiency, cow milk protein allergy, informed consent withdrawal.   
 
Sampling 
Sequential sampling, non probabilistic, until completing the number of required participants.  
Sample size 
To respond to the objective of reduction of at least 12 hours of acute diarrheic disease evolution 
with the administration of zinc, a sample of 36 in each group is required (+20%=44), through the 
calculation of the Student-t sample for independent samples considering 1=2.2, 2=1.7, 
=0.85, =0.05, =0.2, 1 tail, according to the report of Jiang CX, et. al.37 

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To respond to the objective of 15% or more reduction in the number of diarrheic evacuations in 
the group with the administration of zinc, a sample of 25 by group is required (+20%=30), 
through the calculation of Student-t sample for independent samples considering 1=9.06, 
2=10.46, =0.05, =0.05, =0.2, 1 tail, according to the reports of Agarwal, et.al.38  
Finally to respond to the secondary objective of the reduction of acute diarrheic disease relapse 
in children that received zinc in their first clinic picture, considering =0.05, =0.20, Ratio 
Exposed/ Non Exposed=1, Percentage of positive non exposed (basal recurrence) = 20%, risk 
ratio= 0.5 (efficacy), there is a sample required of N=215 by group (+20%=258). 
Do to the above, to give a response to the stated objectives, a sample of 258 children in each 
group is required, considering 20% of loss, meaning a total of 516 children to be included.  
Variable definition 
VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Identification sheet 
ID 
 
Context 
Unique 
alphanumeric 
indicator 
constructed 
with 
a 
consecutive 
number of 4 digits and the initials 
from de first last name, second last 
name and first name.  
Nominal 
polytomous  
0001AAA a9 999ZZZ 
Date of fill 
Context 
Date in which the case study initiates 
and fills the Case Report Format.  
Discrete 
day/MONTH/year 
Attention 
Hospital  
Context 
Medical attention unit where the 
case is identified and put in to the 
study 
Nominal 
1 to k hospitals 
File  
Context 
Number or alphanumeric expression 
corresponding to the hospital file for 
the identification and gathering of 
medical information from the case. 
Nominal 
Alphanumeric, n categories 
individual 
Name minor 
Context 
Complete name of the minor with 
last name 1, last name 2 and 
name(s) 
Nominal 
Open alphanumeric chain; n 
Names 
Sex 
Independent 
Biological condition of the sex that 
the minor belongs to  
Nominal 
dichotomous 
1. Men 
2. Women 
Date of birth 
Context 
Calendar 
date 
registered 
with 
month, day and year in which the 
minor was born 
Quantitative 
discrete 
dd/MMM/yyyy 
Age 
Independent 
Age of the minor in months and days 
turned since the date of birth up to 
the beginning of the case study 
Numeric 
X months, Y days 
Address 
Context 
Physical address where the minor 
and the parents or legal guardians 
can be located, specifying street 
name, exterior number, interior 
number, neighborhood or borough 
or district, zip code, municipality or 
delegation, 
federal 
entity 
and 
particular references.   
Nominal 
Alphanumeric open with n 
categories 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
Page 14 of 33 
 
VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Address 
references 
Context 
Places known that will help as 
reference for the easy localization of 
the patients address.  
Nominal 
Open  
Telephones 
Context 
Telephone numbers in which the 
minor or parents or legal guardians 
could be found, corresponding to 
home telephone, work, cellphones 
or 
of 
family 
members 
or 
acquaintances.  
 
 
 
 
Nominal 
Numeric 
open 
with 
n 
amounts of telephones 
Non pathological Personal Background 
Rooms 
in 
the 
house 
Context 
Number of rooms that the house has 
where the minor lives, taking into 
consideration 
the 
living 
room, 
kitchen and bathrooms.  
Quantitative 
discrete 
0-98 
People living in 
the house  
Context 
Number of people with the minor 
that live on the house.   
Quantitative 
discrete 
1-98 
Mother´s level of 
education 
Context 
Last academic grade the mother was 
in, and if it was completed or not.  
Ordinal  
a. None  
b. 
Elementary  
(complete/incomplete) 
c. 
Middle 
school 
(complete/incomplete) 
d. 
High 
school 
(complete/incomplete) 
e. 
Technical 
(complete/incomplete) 
f. 
Collage 
(complete/incomplete) 
g. 
Undergraduate 
(complete/incomplete) 
h. Unknown. 
Fathers 
level 
education  
Context 
Last academic grade the father was 
in, and if it was completed or not. 
Ordinal 
a. None  
b. 
Elementary  
(complete/incomplete) 
c. 
Middle 
school 
(complete/incomplete) 
d. 
High 
school 
(complete/incomplete) 
e. 
Technical 
(complete/incomplete) 
f. 
College 
(complete/incomplete) 
g. 
Undergraduate 
(complete/incomplete) 
h. Unknown 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Television 
Context 
Existence of a television in the house 
minor lives in.  
Nominal 
polytomous 
1. Yes 
2. No 
99.Does not know 
Refrigerator 
Context 
Existence of a refrigerator in the 
house minor lives in. 
Nominal 
polytomous 
1. Yes 
2. No 
99.Does not know 
Automobile 
Context 
Existence of a automobile in the 
house minor lives in. 
Nominal 
polytomous 
1. Yes 
2. No 
99.Does not know 
Computer 
Context 
Existence of a computer in the house 
minor lives in. 
Nominal 
polytomous 
1. Yes 
2. No 
99.Does not know 
Water 
in 
the 
house 
Context 
Type of water disposition in the 
house the minor lives in 
Nominal 
polytomous 
1. Inside the house 
2. Outside the house but in 
the building, community 
or land. 
3. From a public tap 
4. Well 
5. Pipe or deposit 
6. Not available 
7. Other____________ 
99.Does not know 
Sewage 
Context 
Type of sewer of available in the 
house minor lives in. 
Nominal 
polytomous 
1. Connected to the sewer 
in the street 
2. Connected to a septic pit 
3. Connected to the ground, 
river or lake 
4. No sewage 
99.Does not know 
Breast fed ever  
Independent 
At one point in their lives the minor 
received breast milk feeding. 
Nominal 
polytomous 
1.Yes 
2.No 
99.Does not know 
Age 
maternal 
lactation  
Independent 
Age in months the minor was when 
he finished being breastfed.  
Numeric 
continuous 
Months 0-24 
Still 
being 
breastfed 
Independent 
At the moment of the interview, the 
minor was still breastfed.  
Nominal 
polytomous 
1.Yes 
2.No 
99.Does not know 
Actual feeding 
Independent 
Predominant diet with which the 
minor has been fed at the moment 
of the inclusion. . 
Nominal 
polytomous 
1. Only maternal breast 
2. Dairy formula exclusive 
3. Mix with 
predominant 
maternal breast 
4. Mix with 
predominant 
dairy formula 
5. Mix with 
predominant 
solids 
99.Does not know 

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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Attends daycare 
currently 
Control 
If the child attends daycare regularly  Nominal 
dichotomous 
1.Yes 
2.No 
Time 
attending 
daycare 
Control 
The time since the child attended 
the daycare for the first time up to 
the last day he attended daycare.  
Numeric 
X months, Y days 
Immunization backgrounds 
National Health 
Record 
Context 
If there was a copy of minor´s 
National Health Record attached to 
the Case Report Form  
Nominal 
1=Yes 
2=No 
Dosage 
1 
rotavirus vaccine 
Independent 
If the first dosage of the rotavirus 
vaccine was applied.  
Nominal 
1=Yes 
2=No 
99=Does not know 
Vaccine 
type 
dosage 1 
Independent 
Biological type of the rotavirus 
received at the moment of the first 
dosage 
of 
their 
immunization 
scheme 
Nominal 
1=RotaTeq (RV5) 
2=Rotarix (RV1) 
99=Does not know 
Date of dosage 1 
Independent 
Day, month, year in which the 
patient received their first rotavirus 
vaccine dosage.   
Numeric 
n 
dates 
with 
day/MONTH/year 
Dosage 1 source 
Context 
Source where it was obtained the 
date of application and the type of 
vaccine of rotavirus of dosage 1.  
Nominal 
1=National Health Record 
2=Temporal 
immunization 
proof 
3=Verbal 
4=Nominal 
census 
(immunization unit) 
Immunization 
unit dosage 1 
Context 
Health 
services 
establishments 
where the first dosage of the 
rotavirus vaccine was administered.   
Nominal 
Up to n immunization units 
Dosage 
2 
rotavirus vaccine 
Independent 
If dosage 2 of the rotavirus vaccine 
was applied 
Nominal 
1=Yes 
2=No 
99=Does not know 
Type of vaccine 
dosage 2 
Independent 
Biological type of the rotavirus 
received at the moment of the 
second dosage of their immunization 
scheme 
Nominal 
1=RotaTeq (RV5) 
2=Rotarix (RV1) 
99=Does not know 
Daye dosage 2 
Independent 
Day, month, year in which the 
patient 
received 
their 
second 
rotavirus vaccine dosage.   
Numeric 
n 
dates 
con 
day/MONTH/year 
Dosage 2 source 
Context 
Source where it was obtained the 
date of application and the type of 
vaccine of rotavirus of dosage 2 
Nominal 
1=National Health Record 
2=Temporal 
immunization 
proof 
3=Verbal 
4=Nominal 
census 
(immunization unit) 
Immunization 
unit dosage 2 
Context 
Health 
services 
establishments 
where the second dosage of the 
rotavirus vaccine was administered.   
Nominal 
Up to n immunization units 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Dosage 
3 
rotavirus vaccine 
Independent 
If dosage 3 of the rotavirus vaccine 
was applied 
Nominal 
1=Yes 
2=No 
99=Does not know 
Type of vaccine 
dosage 3 
Independent 
Biological type of the rotavirus 
received at the moment of the third 
dosage 
of 
their 
immunization 
scheme 
Nominal 
1=RotaTeq (RV5) 
99=does not know 
Date dosage 3 
Independent 
Day, month, year in which the 
patient received their third rotavirus 
vaccine dosage 
Numeric 
n 
dates 
with 
day/MONTH/year 
Source dosage 3 
Context 
Source where it was obtained the 
date of application and the type of 
vaccine of rotavirus of dosage 3 
Nominal 
1=National Health Record 
2=Temporal 
Immunization 
proof 
3=Verbal 
4=Nominal 
census 
(Immunization unit) 
Immunization 
unit dosage 3 
Context 
Health 
services 
establishments 
where the third dosage of the 
rotavirus vaccine was administered. 
Nominal 
Up to n immunization units 
Vaccine 
observations 
Context 
If there exist observations about the 
immunization state that up to the 
doctors 
judgment 
must 
be 
registered in the case study.  
Nominal 
Alphanumeric Open 
Personal Pathological backgrounds 
Health problem  
Control 
If the minor´s caregiver refers to a 
health problem.   
Nominal 
dichotomous  
1. Yes  
2. No 
99.Does not know 
Specified health 
problem 
Control 
Detailed description in case there is 
a health problem  
Nominal 
Open 
Hospitalizations 
background due 
to diarrhea  
Control 
Hospitalization appearance due to 
gastroenteritis in their lives without 
taking into accounts the actual, and 
the age of that event.  
Nominal 
1.Yes 
2.No 
99.Does not know 
Age 
at 
the 
moment of the 
previous 
hospitalization 
due to diarrhea 
Control 
In case of having hospitalizations 
background due to diarrhea, indicate 
the age at the moment that 
happened. 
Numeric 
X months 
Hospitalization 
background due 
to pneumonia. 
Control 
Hospitalization appearance due to 
pneumonia 
through 
their 
lives 
without counting the actual, and the 
age in which the event happened.  
Nominal 
1.Yes 
2.No 
99.Does not know 

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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Age 
at 
the 
moment of the 
previous 
hospitalization 
due 
to 
pneumonia 
Control 
In case of having any backgrounds of 
hospitalization due to pneumonia, 
indicate the age at the moment of 
the event.  
Numeric 
X months 
Diarrheic disease 
pictures 
Control 
Number of acute diarrheic disease 
pictures that they have presented in 
the last year.  
Quantitative 
discrete 
1 It is the first episode 
2 two to three pictures 
3 four to five 
4 more than five 
99 Does not know 
Acute respiratory 
infections 
pictures 
Control 
Number 
of 
acute 
respiratory 
infections pictures that they have 
presented in the last year.  
Quantitative 
discrete 
1 It is the first episode 
2 two to three pictures 
3 four to five 
4 more than five 
99 Does not know 
Actual Condition 
Beginning of the 
diarrheic picture 
Control 
Date in which the clinical picture 
with diarrhea initiated. 
Quantitative 
Discrete 
dd / mmm/yyy 
Time 
of 
the 
beginning of the 
diarrheic picture 
Control  
Time of the day in which the clinical 
picture with diarrhea started. 
Quantitative 
Discrete 
Hh:mm 
Days 
with 
diarrhea  
Control 
Quantity of days with diarrhea, from 
the beginning of the episode to the 
medical attention. 
Quantitative 
discrete 
Number 
of 
days 1 to X 
Number 
evacuations/ day 
Control 
Maximum amount of evacuations 
decreased in consistency by day 
Quantitative 
discrete 
1 to X 
Consistency 
of 
evacuations 
Control  
Characteristics quality required in 
terms 
of 
firmness 
and 
stool 
complement.   
Nominal 
polytomous  
1 Pasty 
2 Semi liquid 
3 Liquid 
Characteristics of 
the evacuations 
Control 
Presence of mucus or blood in the 
stool. 
Nominal 
polytomous 
1 No 
2 Only mucus 
3 Only Blood 
4 Mucus and 
blood 
Vomit 
Control 
Presence 
of 
vomit 
in 
the 
gastroenteritis episode. 
Nominal 
dichotomous 
1.Yes 
2.No 
Number of days 
with vomit 
Control 
Quantity of days with vomit, from 
the beginning of the episode up to 
the medical attention.  
Quantitative 
discrete 
1 to X 
Number 
of 
vomits 
Control 
Maximum amount of vomit events a 
day 
Quantitative 
discrete 
1 to X  
Temperature 
Control 
Maximum 
body 
temperature 
registered from the minor during the 
diarrheic episode.  
Continuous 
35.0ºC to 42.0ºC 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
Page 19 of 33 
 
VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Place 
the 
temperature was 
taken.  
Control 
Anatomical place in which the 
thermometer was placed for the 
body temperature measurement.  
Nominal 
polytomous 
1 Rectal 
2 Oral 
3 Armpit 
4 Otaca 
5 Frontal 
Infections 
or 
concomitant 
diseases  
Control 
Presence of concurrent diseases with 
the actual condition.  
Nominal 
dichotomous 
1.Yes 
2.No 
Observations 
actual condition 
Context 
Additional optional descriptive notes 
to the actual episode of intestinal 
intussusception.  
Nominal 
Open alphanumeric chain 
Physical exploration at the moment of the initial assessment 
Weight 
Control 
Corporal weight of the minor in 
kilograms 
determined 
at 
the 
moment of the interview.   
Continuous 
Kilograms  
Height 
Control 
Height of the minor in centimeters 
determined at the moment of the 
interview.  
Continuous 
Centimeters 
General 
condition  
Control 
Clinical 
manifestation 
that 
the 
subject presents in relation with 
their alert state.  
Qualitative 
nominal 
1.Alert, active, smiling o 
crying, 
but 
easily 
comforted. 
2.Thirsty, 
restless 
or 
lethargic but irritable 
when touched.  
3.Sleepy, 
hypotonic, 
cold 
or 
sweaty 
or 
comatose. 
99. Does not know. 
Eyes 
Control 
Presence or absence of sunken eyes 
secondary 
to 
the 
dehydration 
caused by diarrhea.  
Nominal 
polytomous 
1.No changes in the usual 
appearance of the eyes 
(normal ocular tone) 
2.Lightly sunken 
3.Very sunken 
99 Does not know 
Oral mucosa 
Control 
Level of dehydration of the oral and 
tongue mucosa, excluding lips.  
Nominal 
polytomous 
1.Humid 
2.Stiky, with viscose saliva 
3.Dry 
99. Does not know 
Tears 
Control 
Presence of tears during crying of 
the 
minors 
an 
indicator 
of 
dehydrated state.  
Nominal 
polynomial 
1.Present 
2.Scarce 
3.Abstent 
99.Does not know 
Treatment 
Area 
of 
first 
contact 
Control 
Area of the hospital where the minor 
received the first medical attention 
for this episode od diarrhea. 
Nominal 
polytomous 
1.Doctors office 
2.Urgent care 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
Page 20 of 33 
 
VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Hydration plan 
Context 
Type of hydration plan according to 
the WHO that was administered to 
the minor on account of the actual 
gastroenteritis episode.  
Nominal  
1.Plan A 
2. Plan B 
3. Plan C 
4.Other________ 
Hydration 
Treatment  
Control 
The action of receiving a medical 
treatment 
consisting 
on 
the 
reposition of the liquids lost during 
the episodes of diarrhea via enteral 
before going to medical attention.  
Nominal 
polynomial 
1.Ambulatory 
with 
oral 
hydration  
2.Urgent 
care 
with 
oral 
hydration   
3.Urgent 
care 
with 
IV 
hydration 
4. Hospitalized 
Antibiotic 
treatment 
Control 
Presence or absence of the antibiotic 
prescription 
in 
their 
current 
condition. 
Nominal 
dichotomous 
1 Yes 
2 No 
Antibiotic name  
Control 
Type 
of 
antibiotic 
that 
was 
prescribed 
for 
their 
current 
condition.  
Nominal 
Alphanumeric 
Antibiotic dosage  Control 
Antibiotic dosage prescribed  
Nominal 
Alphanumeric 
Days 
of 
treatment 
Control 
Number of days the antibiotic was 
prescribed.   
Quantitative 
discrete 
1 to X 
Antiemetic 
treatment 
Control 
Presence or absence of antiemetic 
prescription 
in 
their 
current 
condition. 
Nominal 
dichotomous 
1 Yes 
2 No 
Name 
of 
antiemetic  
Control 
Type 
of 
antiemetic 
that 
was 
prescribed 
for 
their 
current 
condition. 
Nominal 
Alphanumeric 
Antiemetic 
dosage  
Control 
Dosage of antiemetic that was 
prescribed.  
Nominal 
Alphanumeric 
Days 
of 
antiemetic  
Control 
Number of days that the antiemetic 
was prescribed.   
Quantitative 
discrete 
1 to X 
Antidiarrheal 
treatment  
Control 
Presence 
or 
absence 
of 
the 
antidiarrheal prescription in their 
current condition.  
Nominal 
dichotomous 
1 Yes 
2 No 
Antidiarrheal 
name 
Control 
Type of antidiarrheal that was 
prescribed 
for 
their 
current 
condition.   
Nominal 
Alphanumeric 
Antidiarrheal 
dosage 
Control 
Dosage of the antidiarrheal that was 
prescribed.  
Nominal 
Alphanumeric 
Days 
with 
antidiarrheal  
Control 
Number 
of 
days 
that 
the 
antidiarrheal was prescribed.   
Quantitative 
discrete 
1 to X 
Other treatment  Control 
Presence 
or 
absence 
of 
the 
prescription of other medications for 
their actual condition.  
Nominal 
dichotomous 
1 Yes 
2 No 
Name 
of 
lactobacilli 
Control 
Type 
of 
lactobacilli 
that 
was 
prescribed 
for 
their 
current 
condition.  
Nominal 
Alphanumeric 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Dosage 
of 
lactobacilli 
Control 
Dosage of lactobacilli that was 
prescribed  
Nominal 
Alphanumeric 
Days 
with 
lactobacilli 
Control 
Number of days that the lactobacilli 
was prescribed.  
Quantitative 
discrete 
1 to X 
Name 
of 
the 
other medication 
Control 
 Type of other medication that was 
prescribed 
for 
their 
current 
condition.  
Nominal 
Alphanumeric 
Dosage of the 
other medication 
Control 
Dosage of the other medication that 
was prescribed.  
Nominal 
Alphanumeric 
Days 
with 
the 
other medication 
Control 
Number of days that the other 
medication was prescribed. 
Quantitative 
discrete 
1 to X 
Sample of stool for the laboratory  
Collection of the 
stool sample 
Control 
Collection of the stool sample from 
the current episode was achieved.  
Nominal 
1. Yes  
2. No 
99.Does 
not 
know 
Date sample of 
stool 
Control 
Specification of a day 
in the 
calendar, in which the fecal matter 
was taken.  
Quantitative 
continuous 
Day-month- 
year 
Hour 
of 
recollection 
of 
the stool sample 
Control 
Hour of the day in which the 
recollection 
of 
stool 
samples 
initiated. 
Quantitative 
continuous 
Hour: 
min 
_AM/PM 
Collection 
of 
blood sample  
Control 
The collection of the blood sample 
was achieved. .  
Nominal 
3. Yes 
4. No 
99.Does 
not 
know 
Date 
blood 
sample 
Control 
Specification of a day 
in the 
calendar, in which the blood sample 
was taken. 
Quantitative 
Continuous 
Day-month- 
year 
Hour 
of 
recollection 
of 
blood sample  
Control 
Hour of the day in which the 
recollection of blood sample started. 
Quantitative 
continuous 
Hour: 
min 
_AM/PM 
Observation  
Context 
If there exists observations about 
the sample collection that in the 
physician´s 
judgment 
must 
be 
registered in the case study.   
Nominal 
Alphanumeric open 
Responsible 
physician  
Context 
Name of the researcher physician 
responsible of filling the form of the 
hospital´s Case Report.  
Nominal 
Alphanumeric open 
Tracking  
Date filled 
Context 
Date in which the case study 
initiated and the tracking Form is 
filled.  
Discrete 
Day/MONTH/year 

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Type of tracking 
Context 
Manner in which the parents were 
contacted to perform the tracking. 
Nominal 
Telephone 
Domiciliary 
Inpatient 
Ambulatory 
in 
Sentinel 
Center, 
Other. 
Informant 
Context 
Name of the person that provided 
the information of the minor.  
Nominal 
Alphanumeric 
Relation with the 
minor  
Context 
Relation of the informant with the 
minor.  
Nominal 
Alphanumeric  
ADD 
clinic 
picture evolution  
Control 
ADD clinic picture evolution.  
Nominal  
1 Has gotten worse 
2 No improvement, persist 
the same symptomatology 
3 Has improved 
4 The picture has remitted 
totally 
Medical 
assessment 
Control 
Specify the minor needed new 
medical assessment.  
Nominal 
1 yes, in urgent care 
2 yes, in doctor´s office 
3 no 
Nausea 
Control 
Presence or absence of nausea. 
Nominal 
dichotomous 
1 yes 
2 no 
Fever 
Control 
Presence or absence of fever. 
Nominal 
dichotomous 
1 yes 
2 no 
General 
condition  
Control 
Clinical 
manifestation 
that 
the 
subject presents in relation with 
their alert state.  
Qualitative 
nominal 
1.Alert, active, smiling 
or crying, but is easily 
comforted 
2.Thirsty, 
restless 
or 
lethargic but irritable 
when touched 
3. Drowsy, hypotonic, 
cold 
or 
sweaty 
or 
comatose 
99. Does not know 
Oral tolerance 
Control 
Acceptance of the ingest of food and 
liquids. 
Nominal 
No 
Yes, only liquids 
Yes, liquids and solids 
Tablet 
acceptance 
Control 
Ease for the minor to receive diluted 
tablet.  
Nominal 
Ingested 
with 
no 
problem 
Had difficulty, specify  
Administration of 
the tablet from 
the study 
Control 
Administration of the tablet from the 
study (zinc or placebo). 
Nominal 
Yes 
No 
Medication 
Control 
Medications 
that 
have 
been 
administered in the last 24 hours. 
Nominal 
Alphanumeric 

Protocol Version 2.2, May 22, 2017 
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VARIABLE 
(Index/indicator) 
Type 
OPERATIONAL DEFINITION 
MEASUREMNT 
SCALE 
QUALIFICATION 
Home remedies 
Control 
The presence or absence of home 
remedies administration during the 
current condition.  
Nominal 
Yes 
No  
Number 
evacuations/ day 
Dependent 
Maximum amount of evacuations 
decreased in consistency per day.  
Quantitative 
discrete 
1 to X 
Consistency 
of 
evacuations 
Dependent 
Characteristics quality required in 
terms 
of 
firmness 
and 
stool 
complement. 
Nominal 
polytomous 
1 Pasty 
2 Semiliquid 
3 Liquid 
Quantity 
of 
evacuations 
Dependent 
Quantity of evacuations, in relation 
to the surface that covers in the 
diaper.  
Nominal 
polytomous 
1 Usual 
2 Scarce 
3 Abundant 
Vomit 
Control 
Presence 
of 
vomit 
in 
the 
gastroenteritis episode. 
Nominal 
dichotomous 
1.Yes 
2.No 
Number of days 
with vomit 
Control 
Quantity of days with vomit, from 
the start of the episode up to the 
medical attention.  
Quantitative 
discrete 
1 to X 
Abdominal pain 
Control 
Presence of abdominal pain  
Nominal 
Yes, no 
Laboratory results 
Enteropathogen 
Control 
Isolation od enteropathogens in 
stools. 
Nominal 
Alphanumeric open 
Levels of basal 
Zinc 
Independent 
Concentrations 
of 
zinc 
in 
blood/serum at the beginning of 
study.  
Quantitative 
continuous 
0 to X g/dL 
Time tracking the 
sample 
Control 
or 
independent 
Time in days since the beginning of 
symptoms/start 
of 
the 
treatment/clinical cure/end of the 
supplementation? In which a sample 
of blood is taken for zinc level 
determination.    
Discrete 
Hours 
Levels 
of 
zinc 
tracking  
Dependent 
Blood concentration/ zinc serum in 
g/dl in a time X determined.    
Quantitative 
Continuous  
0 to X g/dL 
Basal albumin 
Control 
Serum concentrations of albumin in 
blood at the beginning of the study.  
Quantitative 
continuous 
0 to X gr/dL 
Hemoglobin 
Control 
Hemoglobin concentration in blood 
at the beginning of the study.  
Quantitative 
continua 
0 to X g/dL 
 
Analysis Plan 
The analysis of the results will be by protocol and by intention to treat. Descriptive statistic will 
be used, with measurements of central tendency and dispersion of the numerical data according 
to the distribution of them. Continuous variables with normal distribution: mean and standard 
deviation. Continuous variables with non-normal distribution: median and inter quartile interval. 
The categorical data will be described with simple frequency, proportions and ratios.  

Protocol Version 2.2, May 22, 2017 
ES Zn-Diarrhea 
Page 24 of 33 
 
The comparison of evolution time of ADD and the number of evacuations will be done through 
the Student t-test for independent groups in case the distribution was normal, or with Mann-
Whitney U test if the distribution was non-normal.  
In case there are non-homogenous factors between groups and that they may be potential 
confounders of the duration of the diarrhea and number of evacuations, there will be included 
in an analysis model of two-tailed variance and/or in a multiple lineal regression model in case 
there is more than one potential confounder. The potential confounders taking data from are: 
a) Attention Hospital 
b) Socioeconomic level 
c) Lactation 
d) Childcare  
e) Vaccine 
f) Previous hospitalizations  
g) Temperature-fever 
h) Dysentery  
i) 
Malnourished 
j) 
Antibiotic 
k) Albumin 
l) 
Hemoglobin 
In case the distribution allows it, a stratified analysis for this variables would be perform.  
For the evaluation of relapse risk the Attributable Risk Fraction, the Reduction of the Fraction of 
Attributable Risk and Absolute Risk Reduction would be estimated with the next formulas:    
Reduction of the fraction of 
attributable risk: 
= 
ADD relapse in control group – ADD relapse in zinc group 
ADD relapse in control group 
 
Absolute risk reduction: 
= 
ADD incidence in control group – ADD incidence in zinc group 
The efficacy of the intervention over the duration of the disease and the number of evacuations 
will be modeled with a general lineal model to adjust by the basal characteristics. A p<0.05 
would be considered significant. 
The efficacy estimators in case the treatment groups are not comparable for any non-
homogenous factor in the randomization and/or associated with relapse, will be adjusted by 
logistic regression model considering the following potential confounders:  
a) Age 
b) Socioeconomic level 
c) Lactation 
d) Childcare 
e) Vaccine 
f) Previous hospitalization 
g) Diarrhea severity 

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The correlation between the increment in the zinc serum concentration posterior to the oral 
treatment for 10 days with 20 mg of zinc with the duration of the ADD picture, the number of 
total evacuations and the relapse rate, will be done as follows:  
Variable 
Test 
Zn concentrations and Duration 
Simple Correlation (parametric or non-parametric) 
Zn concentrations and number of evacuations 
Simple Correlation (Parametric or non-parametric) 
Zn concentrations and relapse 
Student t or Mann-Whitney U  
 
 
Specific process of the study 
Study stages and data recollection.  
Recruitment  
The recruitment and tracking of children will be done in the Sentinel Centers assigned to the 
study. To all the children from 6 to 54 months of age that attend for acute diarrheic disease and 
comply with the selection criteria will be invited to participate in the project. In this first 
intervention there will be an explanation given, of the project and how it will be done (reason 
why the study pretends to be done, objective, inclusion criteria, exclusion, phases of the study 
and anthropometric measurements, dietetics and biochemical as well as the implementation of 
the socioeconomic questionnaire, distribution (randomization) and supplement consumption). It 
will try to clarify every doubt that the mothers present. Once the guardians or parents of the 
child accept to participate the intervention will be assigned randomly and the case study form 
will be filled.  
Assignment of intervention 
The assignment of treatment will be done under the randomization scheme in balanced blocks. 
The children will be assigned randomly to one od 2 treatment groups: one group will receive an 
oral dosage daily of 20 mg for 10 days and the other group will receive placebo in a similar way.   
Baseline Visit 
In each one of the health centers the children will be anthropometrically measured by weight, 
size, height, and arm and head circumference. A trained physician in the matter will perform 
these measurements. Also will obtain a sample of 5 mL of venous blood, with the purpose of 
determining afterwards the concentrations of zinc serum, hemoglobin and albumin. A Pediatric 
Nurse with a wide experience in sample taking from children will obtain the blood sample, and 
the corresponding registration will be done. There will also be a questionnaire applied about the 
characteristics of the house as well as the age, education, marital status, and number of 
domestic appliances like radio, television, blender, etc. In the same appointment, the children 
will be assigned randomly to one of the groups for treatment. A sample of stools will be taken 
from each child within 48 hours after being included. Trained and standardized staff will do all 
the interviews and measurements.  

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Follow-up 
A daily telephone follow-up will be done for the ambulatory patients until the resolution of the 
episode, this is to know the number of evacuations, consistency, and administered treatment 
and strengthen the intervention. For the follow-up of relapses telephone calls will be done 
weekly until completing a maximum of twelve months after the inclusion episode. There will be 
two itinerant groups for domiciliary follow-ups for the failed telephone calls.  Furthermore, the 
caretaker will be provided with a symptoms journal, which they will fill out daily during the time 
of the study.  
A sample of 3 mL of blood will be taken from each child, to determine the control zinc levels. 
The total of participants will be randomized in 6 groups, in which a sample of blood will be taken 
from them, in different moments in the following scheme:  
Group  
1 
2 
3 
4 
5 
6 
Time when the control sample was taken,  
After starting the treatment. 
5 days 
10 days 
20 days 
1 month 
2 months 
3 
months 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Flowchart 
 
 

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Ethical considerations 
Consent 
To the parents or guardians, or to who exercise the parental responsibility of each potential 
participant, information related to the study will be provided in verbal and written form. The 
physician that will serve as Local Researcher in each Sentinel Center will conduct an initial 
invitation to the parents and/or guardians of the minor, for the offspring to participate in the 
study and will read the “Informed Consent Letter” to expose the purpose of the investigation 
and will do a description of the same, pointing out that the study will consist of a 15 to 20 
minutes interview, in taking a blood sample from the minor, and the random allocation to one 
of the study groups. Children will receive the treatment that in the physician´s judgment will be 
more adequate for their disease, age, and clinical state. The intervention group will receive 
supplementation and placebo control, during 10 days. The signature of the parents or guardians 
of the minor should be obtained in a voluntary and free manner in the Informed Consent 
Declaration after assuring that the research objective has been comprehended and doubts have 
been cleared, and before the child is included in the study, if they wish that the minor 
participates. If any parent or guardian does not know how to sign, they will print their 
fingerprints, and another person designated by them will sign on their name. The Informed 
Consent Letter will be handed in duplicate, a copy being for the medical record of the minor in 
the study, and the other one for the parents or guardians. Do to the fact that the participants 
are lactating and to the fact that they are not directly related to the process of information 
collection, an assent request is not appropriate. The people responsible for the minor can reject 
the minor´s participation in the study. If the collaborating researcher is a physician treating a 
candidate to be included, the informed consent must be taken and handed by a third party, this 
way the investigator can still be their treating physician and perform the fill out of the “Case 
Report Form (CRF)”. The foregoing in accordance with the legislation and the established 
recommendations regarding ethics.  
 
 Confidentiality 
With the final purpose of guaranteeing confidentiality and participant´s privacy, the 
investigation documents, as well as the informed consent that contain the names of the 
participants and their parents or caretakers will be kept in drawers locked with key, to which 
only authorized personnel will have access to. More so, the electronic data will be stored in 
protected databases with a code or password, to which only authorized personnel will have 
access. The information of the minor recollected in this study will only be used to the same end. 
 Risk  
The study is classified with major and minimum risk. The fact of receiving placebo is acceptable 
because the zinc supplement is not part of the usual diarrhea handling in Mexico, and in case 
that they get zinc does not represent a risk either because of the dosage that is going to be 
administered, which has been recommended and has proven to be safe in other countries. 
Adverse effects have not been described for this amount of zinc. The amount of blood that is 

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going to be taken from the minor is small, and will be only in two occasions in a year, the 
discomforts that they could present are pain at the moment of puncture or a small bruise that 
will disappear in a few days. The stool sample will be directly recollected from the diaper, so 
their child will not undergo an invasive diagnostic procedure.  
The treatment or any attention provided will be standard or usual, so they will not suffer any 
damage directly related to the investigation.  
Benefits 
There is no immediate benefit for the patient, and there is no payment or financial 
compensation for the minor´s participation in the study. The parents or guardians of the 
participating minor may have access to the information collected from their child for the study, 
if needed. After completing the study, it will not be necessary to inform the results to the 
participants, but any relevant findings related to the health of any particular participant, must 
be informed to the parents or guardians of the same.  
Conflict of interests 
There is no conflict of interest involving researchers. The design, logistics, case and laboratory 
studies, analysis, report writing and publication of results will be in charge of the researchers. 
Biosecurity 
All personnel involve in the study must adopt the preventive measures for their protection 
during the collection, storing, transport and handling of the blood samples; taking into 
consideration the requirements set forth in the general provisions applicable in the matter, in 
particular the Mexican Official Norm for environmental Protection - environmental Health – 
biological-infectious dangerous waste - Handling Classification and Specifications, NOM-087-
ECOL-SSA1-2002. The personnel involved in the study must know that the blood samples 
collected are potentially infectious. 
Inappropriate manipulation of the samples may become a biological risk source for the people 
that 
are 
in 
contact 
with 
them 
or 
for 
the 
environment;  
therefore, it will be important to use the necessary personal protection elements to avoid 
exposure with biological risk, such as: 
 
Ocular protection: glasses or visor mask for the laboratory processing.  
 
Gloves (do not substitutes the regular and appropriate hands hygiene), for the collection 
and handling in the laboratory 
 
Coat, in the collection and handling in the laboratory. 
Sample collection must be performed with the necessary personal protection, in a safe place, 
avoiding surface pollution. As part of the safety measures, all the samples must be tagged with 
the following basic data: 
1. 
Patients ID. 
2. 
Sample type that in every case will be fecal matter. 
3. 
Hour and time of collection. 
4. 
Initials of the person collecting the sample.  

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The time from the sampling to the shipment of the local laboratory for its preparation should be 
as short as possible. 
 
Feasibility 
Sentinel centers considered for the study have been selected based on the installed capacity to 
perform the study. In addition to the fact that the population of children under the age of 5 
years is not so geographically dispersed and easy access to these sites, will ensure adequate 
monitoring of the study. 
The sample analysis will be carried out by the laboratory that counts with the standardized test 
and with strict quality control for the determination of blood analytes.  
The research team counts with a wide experience in coordination and tracking of research 
studies. 
The study counts with a specific funding from UNICEF to accomplish the stated objectives.   
Expected results  
The efficacy evaluation of zinc administration over the duration of the episode and number of 
evacuations in children from 6 to 59 months of age with ADD, will allow to know the usefulness 
of such intervention in the treatment of one of the main causes of morbidity and mortality in 
infant population in our country. A scientific publication will be generated in the indexed 
international magazine, and presentation in conferences and congresses.   
 
 

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