Nutritional Support for Lactating Women and Azithromycin to Infants to Improve Growth Outcomes in Peri-urban Slums of Karachi, Pakistan - Randomized Controlled Trial
Phase: NA
Conditions: Undernutrition
Sponsor: Vital Pakistan Trust

 
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PROTOCOL 
 
Nutritional support for lactating women and Azithromycin for infants to improve growth outcomes in 
the peri-urban slums of Karachi, Pakistan – a Randomized Controlled Trial 
(Mumta LW Trial) 
 
Funding agency: Bill & Melinda Gates Foundation 
 
Grantee: VITAL Pakistan Trust (VPT) 
Investigators at VITAL Pakistan Trust 
Principal Investigator: Yasir Shafiq 
Co-Investigator: Amna Tanweer Yazdani 
 
Sub-Grantee: Department of Pediatrics and Child Health, Aga Khan University 
Principal Investigator: Dr. Fyezah Jehan 
Co-Investigator: Dr. Muhammad Imran Nisar 
Study Site: Rehri Goth, Karachi, Pakistan 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Contents 
1. 
INTRODUCTION 
4 
1.1. 
Background 
4 
1.2. 
Review of Literature and trial rationale 
5 
1.1.1. 
Effect of nutritional supplementation during pregnancy and lactation on birth outcome 
and infant growth 
5 
Justification for using Afzaaish Supplement 
6 
1.1.2. 
Effect of antibiotic prophylaxis for infant 
7 
Justification for using Azithromycin 
7 
1.2. 
Rationale for the trials 
8 
2. 
PROJECT STRATEGY AND APPROACH 
9 
3. 
STUDY SETTING AND SITE DESCRIPTION 
10 
4. 
OBJECTIVES 
11 
4.1. 
Primary objective 
11 
4.2. 
Secondary objective 
11 
5. 
HYPOTHESES 
12 
6. 
METHODOLOGY 
13 
6.1. 
Study Design 
13 
6.2. 
Study Population 
13 
6.3. 
Inclusion and exclusion criteria 
13 
Inclusion Criteria 
13 
Exclusion Criteria 
13 
6.4. 
Interventions 
14 
6.4.1. 
Nutritional Supplement 
14 
6.4.2. 
Antibiotic Azithromycin 
18 
6.4.3. 
Standard Counseling and Referral System for Lactating Women 
18 
6.5. 
Enrollment of Lactating Women 
18 
6.6. 
Informed consent procedure for trial recruitment 
19 
6.7. 
Randomization and Allocation Concealment 
19 
6.8. 
Blinding of Outcome Assessor 
20 
6.9. 
Study Drug Storage, Dispensing and Administration 
20 
Afzaaish Supplement 
20 

 
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Azithromycin 
20 
6.10. 
Assessment and Recording Data on Study Participants 
21 
6.11. 
Laboratory sampling for study purpose only 
22 
6.12. 
Follow ups 
22 
7. OUTCOME ASSESSMENT 
24 
7.1. 
Primary Outcome 
24 
7.2. 
Secondary Outcomes 
24 
Other Pre-specified Outcomes 
24 
7.3. 
Trial procedures for outcome assessment 
25 
8. 
ADVERSE OUTCOMES 
26 
8.1. 
Afzaaish supplement related – lactating woman only 
26 
8.2. 
Azithromycin related – Infant  only 
28 
9. 
COMPLIANCE WITH AFZAAISH SUPPLEMENT AND AZITHROMYCIN 
30 
10. 
STATISTICAL ANALYSIS 
31 
Primary 
31 
Secondary objective 
31 
11. 
SAMPLE SIZE CALCULATIONS & POWER ANALYSIS 
36 
12. 
REFRENCES 
37 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1. INTRODUCTION 
1.1. 
Background 
Maternal undernutrition has a critical role in etiology of poor perinatal outcomes. (1) Low birth weight 
(LBW) babies being the most crucial among those with poor outcomes, contributes to 60-80% of all 
neonatal deaths, and (2) impacts nearly 20 million newborns. It is estimated that around 23.3 million 
newborns are also small for gestational age (SGA), {Lee, 2017 #3} (3) and nearly 18.6% of the global SGA 
births are attributed to maternal undernutrition during pregnancy. (4) In low middle income countries 
(LMIC), SGA alone contributes to 32 million cases of stunting. (5) Poor pre-pregnancy nutrition status (low 
body mass index), inadequate weight gain during pregnancy, as well as macronutrient and micronutrient 
deficiencies can lead to poor fetal growth, especially in low income settings. (6) Further, the poor 
nutritional status of these women, which also translates to undernutrition during the lactation phase, 
affects the nutritional status of newborns and infants. (7) Nearly 45% of infant deaths stem from poor 
nutritional gain during the early months of life and underlying wasting and stunting. (7) This also attributes 
to low quality of breastfeed in spite of exclusivity and impacts duration of breastfeeding resulting in 
malnourished infants even with exclusive breastfeeding.  
In Pakistan, nearly 58% of households are food insecure or suffering from starvation. (8) Great disparities 
exist between urban-rural and within urban disadvantaged populations living in the poorest of slums. In 
Sindh province alone, 72% of households are food insecure and 50% are with moderate to severe hunger. 
(8) Around 18% of married woman of reproductive age in Pakistan are underweight based on body mass 
index (BMI) and also deficient of different micronutrients. For example, 41-42% of women are Vitamin A 
and Zinc deficient. (8) This directly impacts childhood stunting, wasting and underweight prevalence, 
which among under-five children in Pakistan is around 44%, 15% and 31%, respectively.  (8) Furthermore, 
approximately 26% of babies have low birth weight and around 36% babies are SGA in Pakistan, (3) and 
these children are at the highest risk of developing moderate to severe stunting or wasting. This mandates 
the need for exploring the role of nutritional supplements in mothers on outcomes such as length velocity 
over the age of six months.  

 
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1.2. 
Review of Literature and trial rationale 
1.1.1. Effect of nutritional supplementation during pregnancy and lactation on birth outcome and 
infant growth 
To address the issue of malnutrition among pregnant and lactating women (PLW) and resultant 
malnutrition among infants, the World Health Organization Antenatal Care (WHO ANC) guidelines and 
National Guidelines for Community-based Management of Acute Malnutrition (CMAM) recommend the 
use of fortified balanced energy-protein supplements during pregnancy and lactation. Most importantly, 
the nutrient needs of infants during lactation depend primarily on the quality of breast milk which the 
infant is receiving from the mother, i.e. both the volume and composition of milk produced (quality of 
breast milk -macro- and micro-nutrients) are important. The quality of breast milk is very much linked to 
the mother's initial nutrient needs, nutritional intake, and nutritional status. Until recently, the WHO ANC 
guidelines had no recommendations on the use of these supplements in food insecure and 
undernourished settings, however, the new shift in policy provides an opportunity to generate evidence 
from developing countries to support the use of fortified balanced energy-protein supplements during 
the early lactation phase (0-6 months of infant’s life) in order to see the impact on the infant’s growth. 
The ideal product would be one which is indigenously produced, fulfills dietary needs, fills energy/nutrient 
gaps, is palatable and is free of side effects. It is of importance that the supplement used shows benefit in 
averting post-neonatal outcomes and has a strong positive impact on growth and development. 
Additionally, there is also a strong debate on the impact of improved human gut microbiota (lactating 
mother and infant) on improved growth outcome. The type of diet consumed by the mother may have an 
impact on the gut microbiota of the infant, which may affect absorption of the macro- as well as micro-
nutrients that the baby receives from the breast milk. Although the understanding of the microbiota 
concept is relatively new, current knowledge strongly suggests that the composition of microbiota has 
profound impact on the health status of humans, especially children. Diet plays a pivotal role in microbiota 
composition, and dietary types and patterns are linked with distinct combinations of bacteria in the 
intestine. Recently, the role of a chickpea based, balanced-energy protein supplement is under discussion, 
which may have great potential to improve gut microbiota. However, such supplements are newly 
available in Pakistan, with limited evidence available from the efficacy and effectiveness view point.  
Available literature evaluating the evidence of Lipid-based Nutrient Supplements (LNS) in lactating women 
studied the efficacy of a small quantity of LNS (20 g/day with an energy of 118 kcal and protein of 2.6 
g/day) provided during pregnancy, lactation and infancy and looked at the effect at 18 months of age. (13) 
A 6 months follow-up  analysis of groups based on the intended supplement showed Mean ± SD of Length-

 
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for-age z score of 0.62 ± 1.04, -0.78 ± 0.96, and -0.72 ± 1.03, for groups that were receiving daily Iron-
folate (IFA) (pregnancy only), multiple micronutrient (MMN) (pregnancy and lactation), and LNS 
(pregnancy, lactation, and infancy) supplementation respectively. (13) Further, in a similar trial, LNS (20 
g/day with energy of 118 kcal and protein of 2.6 g/day) was given to women during pregnancy and 
continued for 6 months after delivery, and the same infant LNS from 6 to 18 month of age with weaning 
diet. The hypothesis was that those infants who were in the LNS group will have a higher mean length at 
18 months than the group where the mother received either iron or folic acid (IFA) during pregnancy only 
or Multiple Micronutrient (MMN) supplementation during pregnancy and lactation and the children who 
did not receive LNSs. This trial showed that at 18 months, the mean length showed an insignificant 
difference of 77.0, 76.9, and 76.8 cm (P value 0.90) in the IFA, MMN, and LNS groups respectively, and the 
prevalence of stunting was reported as 32.7%, 35.6%, and 37.9% (P value 0.54), respectively in all three 
arms. No intergroup differences were found in the mean weight, head circumference, or mid-upper arm 
circumference or the proportions with low z scores for these variables (P > 0.05). (14) Furthermore, 
another trial (Lanou H et al. 2014) looked at the impact of prenatal supplementation with LNS (72 gm/day 
with protein of 14.7 gm per sachet, 372 Kcal) compared to MMN at postnatal infant growth (until 1 year), 
there were no significant differences  reported in linear growth velocity after birth. (15)  
Justification for using Afzaaish Supplement 
The new paradigm shift has strongly suggested the use of balanced protein energy supplementation 
(supplements in which protein provides less than 25% of the total energy content) to the undernourished. 
Use of such supplements by PLW may promote gestational weight gain, improve pregnancy outcomes and 
infant growth. (16) A variety of balanced energy-protein supplements are available, based on quality and 
type of protein, and its acceptability and feasibility for use that have shown varying effects. (17, 18).  LNS 
used in different trials over the period of time are different from Afzaaish product proposed in this trial 
with respect to dose and composition. The supplement used in the Ghana study was designed specifically 
to provide micronutrients for a small amount of energy of about 118 kcal/day not really aiming to fill an 
energy gap, but rather to provide 4 macro minerals (Calcium, Potassium, Phosphorus and Magnesium) 
along with micronutrients and high quality protein of 2.6 gm/day in the amount of a 20 gm sachet per 
day. (13) The product was developed by International Lipid-Based Nutrient Supplements (iLiNS), following 
initial work in Malawi and Ghana. (19-21)  
Until recently, in the context of Pakistan, experience with such ready-to-use formulation/supplements has 
been limited and in many cases the acceptability, efficacy and effectiveness of these products have not 
been properly studied to understand the contextual needs. Furthermore, many of the previously available 

 
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and tested products have low protein content, which has recently been recommended by policy actors. A 
product which provides approximately 16-20 grams of protein per day to PLW may produce a good impact 
on infants’ growth outcomes. Even though there has been progress in the formulation of better nutrition 
supplements that are scalable for integration in MNCH and nutrition programs, evidence for their impact 
on birth outcomes and other postnatal growth effects on infants in LMIC is deficient. ‘Afzaaish’, a high 
protein, balanced protein-energy fortified ready-to-use supplement, is the main intervention in the 
proposed study. It is specifically designed for PLW to provide additional calories and it is locally produced 
in Pakistan. Afzaaish is a lipid-based and fortified nutrition supplement in the form of a semi-solid paste, 
with palatable taste which is likely to be accepted by the local population. Therefore, based on the need 
for evidence in the synthesis of new robust policies, this trial is envisioning the use of the ‘Afzaaish’ 
product among lactating women, which is high protein, balanced-energy, and has a chickpea base. This 
could impact the quality of breast milk and improve the nutritional status of mothers and infants, and may 
also have an impact in improving gut microbiota.  
1.1.2. Effect of antibiotic prophylaxis for infant 
Furthermore, there is also strong debate on the prophylactic use of antibiotics (especially the use of 
Azithromycin AZM) in averting the risk of stunting and underweight, although there is little evidence in 
support. The recent studies showed significant impact on the reduction of all causes of under-five 
mortality, but were unable to demonstrate a statistically significant difference in stunting, underweight, 
and low MUAC of children. The role of antibiotics on child growth and nutrition remains unclear, but the 
authors suggested larger studies and longitudinal trials may help determine any association. (9-12) The 
use and safety profile of AZM in infants is also well documented. A systematic review showed that AZM is 
the most common antibiotic used to treat several pediatric infections, including bronchopulmonary 
dysplasia (BPD). (22) Some side effects noted with its use include diarrhea, abdominal pain and vomiting. 
There is a potential side effect of infantile hypertrophic pyloric stenosis (HIPS). According to one author, 
the risk is high in the neonatal age and still needs to be further investigated. (23) Its use beyond the 
neonatal age is considered to be safe. (22, 23) 
Justification for using Azithromycin 
Literature has provided compelling evidence that AZM is safe for use in young infants and children (10, 
23). Use of oral AZM on a mass level to control overall mortality in children living in rural regions has been 
found efficacious against respiratory diseases, diarrhea and malaria. (11, 12). In another study, giving a 
single oral prophylactic dose of AZM dramatically reduced the prevalence of infections in endemic areas. 

 
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(10) However, the treatment may have unintended consequences including possible adverse effects or 
resistance to antibiotics, but so far no data suggests resistance to a single oral prophylactic dose of AZM. 
(26) Furthermore, the mechanism of action of AZM is multidirectional and it plays a vital role in reducing 
inflammation because of its anti-inflammatory properties. (27, 28) Oral administration of AZM has also 
been found to improve the diversity of gut flora, improve immunity (29) and prevent the risk of infection 
among children. However, these theories have not been proven yet and further evidence to support 
prophylactic use of AZM among infants to promote infant growth is needed through large trials. Also, 
evidence from the combined effect of nutrition supplementation to lactating woman and prophylactic 
dose of AZM to infants is not yet available to see the overall impact on infants’ growth outcomes.  
1.2. 
Rationale for the trials  
Robust evidence is needed via a field trial in the local context to evaluate the efficacy and effectiveness 
of the locally-produced, balanced energy-protein Afzaaish supplement alone or in combination with 
prophylaxis dose of AZM to infants on maternal and infant growth outcomes in low-income and food 
insecure settings. This could help to draw inferences for larger public health policy-making. This 
investment is specifically aiming to look at what impact a newly formulated nutritional supplement for 
pregnant and lactating women (PLW) can have on improving birth outcomes and as well as its potential 
to reduce wasting, stunting and underweight in infants. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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2. PROJECT STRATEGY AND APPROACH 
With the above background and rationale, we proposed a trial to address the impact of nutritional 
supplementation on lactating women by using a fortified, balanced energy-protein supplement alone in 
women or in combination with prophylactic use of azithromycin to infants. The trial will be conducted in 
peri-urban low-income areas of Karachi (Rehri Goth, Bhains Colony and Ali Akber Shah). The trial will be 
conducted in parallel to gain efficiency and obtain results on postnatal growth outcomes within a short 
timeline. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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3. STUDY SETTING AND SITE DESCRIPTION 
The trial will be conducted at Rehri Goth, Bhains Colony and Ali Akber Shah, Karachi. These are 
contiguously located impoverished peri-urban coastal slums with a population of approximately 250,000 
residents based on the census conducted in 2017. These sites have an annual birth cohort of around 5000 
each year. Fishing is the most common occupation (~30%). The population is multi-ethnic with Sindhi, 
Pashtun, Punjabi, Bengali, and Urdu-speaking groups.  At our proposed site of work, a partner organization 
(Aga Khan University Hospital, AKUH) has a long-standing demographic surveillance system (DSS) in place. 
This includes 2 monthly Married Women Surveillance Registration (MWSR) rounds, which includes all 
women of 13- 49 years of age living within the designated area. During the surveillance rounds, the vitals 
of the women, including mortality, pregnancy status, and movement in and out the area, as well as the 
status of under 5 children, are recorded.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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4. OBJECTIVES 
4.1. 
Primary objective 
a) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product for at least 6 months during lactation and exclusive breastfeeding, in 
improving length velocity as the primary outcome. 
b) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product for at least 6 months during lactation and exclusive breastfeeding along with 
oral Azithromycin at 42 days of age to the baby as a single dose in improving length velocity as 
the primary outcome. 
4.2. 
Secondary objective 
a) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product for at least 6 months during lactation and exclusive breastfeeding in 
improving breast milk composition over the period of six months and weight velocity, length-for-
age, weight-for-length, and weight-for-age Z scores as secondary outcome at 6 months of age as 
compared to the standard of care. 
b) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product for at least 6 months during lactation and exclusive breastfeeding in 
improving breast milk composition over the period of six months along with oral Azithromycin at 
42 days of age to the baby as a single dose, and weight velocity, length-for-age, weight-for-length, 
and weight-for-age Z scores as secondary outcome at 6 months of age as compared to the 
standard of care. 
 
 
 
 
 
 
 
 
 
 

 
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5. HYPOTHESES 
Length velocity at 6 months of infant’s age 
Ha = Mean difference in length velocity of >0.12cm/month to look at comparisons between multiple arms 
Ho = Mean difference in length velocity of ≤ 0.12cm/month to look at comparisons between multiple arms 
Growth velocity at 6 months of infant’s age 
Ha = Mean difference in growth velocity of >0.4 gram/kg/day to look at comparisons between multiple 
arms 
Ho = Mean difference in growth velocity of ≤ 0.4 gram/kg/day to look at comparisons between multiple 
arms 
Length-for-Age Z-score (LAZ) at 6 months of infant’s age 
Ha = Mean difference in LAZM >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in LAZM ≤0.5 to look at comparisons between multiple arms 
Weight-for-Length Z-score (WLZ) at 6 months of infant’s age 
Ha = Mean difference in WLZ >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in WAZM ≤0.5 to look at comparisons between multiple arms 
Weight-for-Age Z-score of (WAZ) at 6 months of infant’s age 
Ha = Mean difference in WAZM >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in WAZM ≤0.5 to look at comparisons between multiple arms 
 

 
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6. METHODOLOGY 
6.1. 
Study Design 
A randomized, controlled, open-label, community based trial to assess the impact of the provision of 
nutritional intervention to lactating women from 0-6 months post birth outcome and one prophylactic 
dose of AZM to infants on the infants’ growth outcome. Individuals responsible for outcome assessment 
(the monthly anthropometry) will be kept blinded from the participants' information randomized in each 
arm.  
6.2. 
Study Population 
Lactating women and their babies until 6 months of age, residing in low-resource settings 
6.3. 
Inclusion and exclusion criteria  
Inclusion Criteria 
1. Live birth, captured within 0-6 days 
2. MUAC of mother < 23.0 cm 
3. Permanent resident of the catchment area, i.e. resident since last the 6 month at least (not 
temporary migrant or guest/relative) 
4. Confirmed intention to exclusively breastfeed their child for at least 6 months of age 
5. Child continues to stay with mother for the next six months 
6. Provision of written informed consent 
Exclusion Criteria 
1. Working and employed lactating women staying away from the baby and the house, as exclusive 
breastfeeding is a prerequisite for enrollment in this trial 
2. VLBW i.e. < 1500 gram 
3. A mother who is unable to breastfeed the child due to certain medical conditions.  
4. Child with congenital abnormalities (example: Cleft palate/lip, hydrocephalus, microcephalus, 
intestinal obstruction, etc.) or other serious illness at the time of enrollment, which was identified 
by the study physician (suspected sepsis for example) 
5. Previously enrolled in this trial or PW trial 
6. Known allergy to food items like, peanut, lentils, milk etc.  
 
 
 
 

 
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6.4. 
Interventions 
 
Arm A 
(Control) 
Arm B 
(Afzaaish Only) 
Arm C 
(Afzaaish + AZ) 
 
Afzaaish 
Supplement 
Not Applicable 
Two sachets per day from  the 
day of enrollment to 6 months 
A weekly supply of sachets 
will be given to each LW 
Two sachets per day from the 
day of enrollment to 6 months 
A weekly supply of sachets 
will be given to each LW 
 
Azithromycin 
Not Applicable 
Not Applicable 
 
To be given to infants of LW 
Infant dose: 20mg/kg at 42 
days of age after birth 
 
Standard 
Counseling 
As mentioned below 
(5.4.3) 
As mentioned below 
(5.4.3) 
As mentioned below 
(5.4.3) 
 
6.4.1. Nutritional Supplement 
‘Afzaaish’ is the main intervention in the proposed study. Locally produced in Pakistan, it is specifically 
designed for PW and LW to provide additional calories. Afzaaish is a fortified, semi-solid, lipid-based 
nutrition supplement which is available as a ready-to-use paste. As per information provided by the 
manufacturer, table 1 provides the product specifications: 
Table 1: Afzaaish Supplement Product Specifications 
S. No. 
Ingredients 
% 
Dry 
Weight 
1 
Roasted chickpeas 
21.2 
2 
Skimmed milk powder 
20 
3 
Soybean oil 
15 
4 
Roasted peanuts 
13 
5 
Sugar 
10 
6 
Palmolein oil 
9 
7 
Roasted yellow lentils 
4 
8 
Premix – vitamin and minerals (approximate premix incorporation rate = 34 
per metric ton) 
3.4 
9 
Hydrogenated vegetable fat as stabilizer 
2 
10 
Maltodextrin (14-18 dextrose equivalent) 
2 
11 
Emulsifier 
0.34 
12 
Antioxidant 
0.068 
 
According to the product information provided by the local manufacturer, the detailed range of nutrients 
and nutritional values per 100 gram of the finished product is mentioned below (these ranges are provided 
by the manufacturer and mentioned on the label of the product): 
i. 
Energy = 510-560 Kcal 

 
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ii. 
Protein = 11-16 gram   
iii. 
Skimmed milk powder protein = 3.6 grams 
iv. 
Fat = 26-36 grams 
v. 
Omega 3 fatty acids = 1.80 grams 
vi. 
Omega 6 fatty acids = 6.10 grams 
vii. 
Added sugars = 10 grams 
Serving size is 75 grams according to the label. The product has a shelf life of 18 months with respect to 
the climate of Pakistan and 24 months when stored up to 300 C at 65% relative humidity.  Detailed product 
specification with recommended daily requirement is provided in table 2 below: 

 
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Table 2: Afzaaish Supplement- Recommended Daily Requirement 
IOM DRIs/RDAs/AIs and FAO/WHO RNIs for Pregnant and Lactating Women -Macronutrients and Micronutrients 
 
Micronutrients - 
Minimum and 
Maximum by the 
expert convening 
Afzaaish product - Locally produced ready-to-use 
supplement 
IOM Energy DRIs for 19-30 years 
FAO/WHO RNIs for 19-30 years 
Pregnant women (additional kcal/day) 
Energy (Kcal) 
First Trimester 
Second 
Trimester 
Third Trimester 
First 
Trimester 
Second 
Trimester 
Third 
Trimester 
 
 
Mini
m-um 
/ 
Targe
t 
(EAR) 
 
 
Maxim-
um 
(RDA) 
Min 
per 
100 
gm 
Label 
per 100 
gm 
Label per 75 
gm sachet 
(One serving 
minimum) 
Expected energy 
that dose of 2 
sachets per 
enrolled PW/LW 
will provide (150 
gm per day) 
(Approx.) 
None 
340 
452 
85.0 
285.0 
475 
Total 
energ
y 250 
Total 
energy 
500 
510 
510-560 
400 
800 
Lactating women (additional kcal/day) 
Macronutrients and Micronutrients 
Less than 6 months of infant age 
330 
505 
 
Requirement 
Pregnant 
Lactating 
Pregnant 
Lactating 
Maximum 10% of 
total energy 
Carbohydrate (g) 
RDA 
175 
210 
 
 
Protein (g) 
RDA 
71 
71 
Additional 1, 9 
and 
31 g in 1st, 2nd 
and 
3rd trimester 
Range of additional 
14.3 to 16.2 g in 1st 
6months 
 
 
14 
 
18 
11 
11-16 
10.5 
21 
Lipid total (g) 
- 
- 
- 
 
 
10% of 
total 
energy 
60% 
of 
total 
ener
gy 
26 
24-36 
24 
48 
Linoleic acid (g) 
AI 
13.0 
13.0 
 
 
 
 
2.6 
2.6-6.10 
2.6 
5.2 
Linolenic acid (g 
AI 
1.4 
1.3 
 
 
1.3 
 
0.3 
0.3-1.8 
0.3 
0.6 
Fiber (g) 
AI 
28 
29 
 
 
 
 
 
 
 
 
Vitamin A (μg RE) 
RDA 
770 
1300 
RNI:370 
Safe intake:800 
RNI: 
50, Safe in 
take:850 
550 
770 
550 
550 
4 
2 
 
24 
Vitamin D (μg) 
RDA 
 
5 
15 
15 
10 
15 
1 
15 
11 
2 
22.4 
Vitamin E (mg) 
RDA 
15 
 
9 
NR 
NR 
16 
19 
16 
16 
12 
24 
Vitamin K (μg) 
AI 
90 
90 
55 
55 
72 
90 
27 
27 
20.2 
40.4 
Thiamin (mg) 
RDA 
1. 
1.4 
RNI:1.4 
RNI:1.5 
1.2 
1.4 
1 
1 
0.75 
1.5 
Riboflavin (mg) 
RDA 
1.4 
1 
1.4 
 
1.3 
1.6 
2 
2.1 
1.57 
3.14 

 
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6 
.6 
1 
Niacin (mg) 
 
DA 
1 
1 
18 
17 
14 
18 
 
3 
13 
9.75 
19.5 
Vitamin B6 (mg) 
RDA 
1.9 
2.0 
1.9 
 
.0 
1.7 
2 
1.8 
1.8 
1.35 
2.7 
Folate (μg) 
 
DA 
600 
500 
EAR:520, RNI:600 
EAR:450, R 
NI:500 
400 
600 
33 
330 
2 
7 
494 
Vitamin B12 (μg) 
RDA 
2.6 
2.8 
EAR:2. 
, RNI:2.6 
EAR:2.4 
RNI:2.8 
2.4 
2.8 
2. 
2 
7 
2 
4 
Vitamin C (mg) 
RDA 
85 
120 
55 
70 
100 
120 
60 
6 
45 
90 
Calcium (mg) 
RDA 
1000 
1000 
1200 (Last 
Trimester) 
 
000 
500 
1000 
535 
535 
400 
800 
Iron (mg) 
RDA 
27 
29 
 
10 to 30 
22 
27 
10 
10 
7.5 
15 
Zinc (mg) 
RD 
11 
12 
5.5, 7, 10 
(1st, 2nd 3rd 
trimester) 
9.5, 8.8 
(0-3, 3-6 months) 
15 
20 
11 
11 
8.2 
16.4 
Iodine (μg) 
RDA 
220 
2 
0 
200 
200 
209 
290 
 
00 
100 
75 
150 
Biotin (μg) 
AI 
30 
35 
30 
35 
28 
35 
60 
60 
 
5 
90 
Pantothenic acid 
(mg) 
AI 
6 
7 
6 
7 
5.6 
7 
4 
4 
3 
6 
Choline (mg) 
AI 
450 
550 
 
 
220 
550 
 
 
 
0 
Phosphorus (mg) 
RDA 
700 
700 
 
 
300 
700 
450 
450 
337 
674 
Magnesium (mg) 
RDA 
350 
310 
220 for females> 
19, NR 
220 for females> 
19, 
NR 
145 
350 
150 
150 
112 
224 
Manganese (mg) 
RDA 
2.6 
2.6 
 
 
2.1 
2.6 
1.2 
1.2 
0.9 
1.8 
Copper (μg) 
RDA 
1000 
1300 
 
 
1 
1.3 
140
0 
1000 
1000 
2000 
Selenium-um (μg) 
RDA 
60 
70 
28 (2nd trimester 
) 
30 (3rd trimester) 
35 (0-6 months post-
partum) 
60 
70 
20 
20 
15 
30 
Potassium-um (g) 
AI 
4.7 
5.1 
 
 
2 
5.1 
0.9 
0.9 
 
.675 
1.35 
IOM = Institute of Medicine 
FAO = Food and Agriculture Organization of the United Nations 
WHO =World Health Organization 
DRI = Dietary Reference Intake 
AI = Adequate intake 
EAR = Estimated Average Requirements 
RDA = Recommended Dietary Allowance 
RNI = Reference Nutrient Intake 

 
Page 18 of 38 
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6.4.2. Antibiotic Azithromycin 
Azithromycin is an azalide antibiotic with a good safety profile and it can be safely used in infants. 
Azithromycin will be given to infants at 42 days of age after birth at a standard dose of 20mg/kg as a single 
oral dose in the form of an oral suspension. Use of oral Azithromycin can be administered to neonates as 
a single dose of 20mg/kg without the report of any adverse events and is thus considered safe. (23)  
6.4.3. Standard Counseling and Referral System for Lactating Women  
Once LWs are confirmed to be within the eligibility criteria, they will be given routine ANC counseling, as 
well as nutritional and exclusive breastfeeding counseling, immunization services, primary health care 
services for newborn/infants and referrals in case of the newborn/infant when and where required. 
Standard nutritional and exclusive breastfeeding counseling is an essential component of the WHO MNCH 
program. The strategy focuses on: 
i. 
Initiation of breastfeeding within the first hour of life 
ii. 
Exclusive breastfeeding – that is, the infant only receives breast milk without any additional 
food or drink, not even water 
iii. 
Breastfeeding on demand – that is, as often as the child wants, day and night 
iv. 
No use of bottles, teats or pacifiers 
v. 
Promoting a healthy diet by increasing the diversity and amount of foods consumed 
vi. 
Promoting adequate weight gain through sufficient and balanced protein and energy intake, 
through the consumption of a variety of foods, including green vegetables, meat, liver, fish, 
beans, lentils, dairy products and fruits. 
Apart from counseling and follow-ups, LW in Arm A will be provided a small package once at the time of 
enrollment in good faith for contributing their time for this trial. Furthermore, nutritional counseling and 
provision of high quality standard of care will be the same for all arms, as per WHO guidelines. Along with 
this, all groups will be routinely followed for assessing danger signs and to check the commitment with 
continuation of exclusive breastfeeding habits. 
6.5. 
Enrollment of Lactating Women 
LW will be enrolled in the project based on case definition (inclusion criteria) and eligibility assessment. 
The research team will identify potentially eligible candidates during pregnancy and wait till the birth 
outcome to visit the household within 0-6 days of live birth to confirm eligibility. When the birth outcome 
is reported, following eligibility assessments, LWs will be enrolled in the trial after all key procedures of 
consent and randomization. Written voluntary informed consent in the presence of a witness will be taken 

 
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Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
to ensure voluntary participation in the trial. After consent, LW will be randomized following the defined 
sampling strategy.  
Related to notification of birth outcomes, VPT has a very robust surveillance system through which 
community health workers will notify the birth outcomes in the existing system through household PW 
follow-ups designed for this purpose. Moreover, for this trial to create liaison with the community, a very 
successful phone call system has been established through which the community is connected with a 
designated health worker 24/7. Therefore, any woman who delivers at any other facility or at home can 
also notify the study team though a phone call. An incentive has been established to encourage the family 
members to call within the time limit. 
Once an LW with MUAC <23.0 cm and birth outcome captured within 0-6 days is identified, the LW will 
be counseled with regard to the trial procedure and will be given a time of 24-48 hours to discuss it with 
family members. Where required, randomization teams will visit the household to develop liaison with 
the family and explain the procedure. Once the woman and family demonstrate voluntary willingness, 
informed consent will be explained once again before the final signing of the consent in the presence of 
a witness. Following this, she will be randomized in one of the three arms according to the trial strategy.  
6.6. 
Informed consent procedure for trial recruitment 
LW fulfilling the case definition and inclusion criteria are required to sign informed consent voluntarily to 
participate in the trial. A verbal consent (informal permission to develop rapport) of the husband or any 
other household member will also be sought in order to avoid dropping out of the trial at a later stage. 
Consent for study participation will be obtained by the research assistant in the presence of a community 
health worker and will involve detailed verbal communication in the study participants’ native language 
to ensure comprehension of the trial and study procedures. Illiterate guardians will be requested for 
thumb impression while literate guardians will be requested to sign the consent form. All the enrolled 
participants in all of the groups will get a copy of the consent form duly signed by the study officials. The 
informed consent procedure will be conducted in the local languages.  
6.7. 
Randomization and Allocation Concealment 
The block randomization technique will be used in this trial and works by randomizing participants within 
blocks such that equal numbers are assigned to each treatment. Blocks will be of varying size as 3, 6, and 
9 in a ratio of 1:1:1. In each arm, 319 LWs will be randomized. The randomization list will be generated 
independently by a data management unit not involved in recruitment, outcome or any other aspect of 
the study. Following the list, the sealed envelopes will be prepared. All those LWs who consent to 
participate in the study will be randomized into the respective arm by the randomization team. For 

 
Page 20 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
randomization in Afzaaish LW trial, the sealed envelope technique will be used. Envelopes will be placed 
in a box file that will be carried by the randomization team. After taking consent from the LW, the research 
staff will pick the top most envelope from the file in a sequential manner. This allows them to follow the 
implemented strategy of ‘sequence within a block’ as well as ‘block sequence’. The opening of an envelope 
will reveal the unique randomization ID with the allotted treatment arm the LW would be enrolled in. 
6.8. 
Blinding of Outcome Assessor 
There will be two separate assessment teams involved in this trial. Outcome assessment team (Anthro 
team) will include the research staff that will be responsible for conducting monthly assessments of the 
outcomes. This team will remain blinded with respect to the allocation of interventions and will be 
restricted with their inquiries. Their work will be limited to anthropometry and outcome assessments 
according to the designed schedule. 
Team 2 (Follow-up Team) will comprise of the research staff who will be responsible for compliance checks 
and distribution of Afzaaish sachets on a routine basis, as well as for administration of the Azithromycin 
dose to the infants and food frequency checks. Technically, this team cannot be blinded to the 
intervention allocation. Also, they will be involved in conducting regular counseling sessions on exclusive 
breastfeeding and healthy diet. This team will also be restricted to follow the standard counseling protocol 
for all the enrolled LW in the trial.  
6.9. 
Study Drug Storage, Dispensing and Administration 
Afzaaish Supplement 
LWs allocated to arms B and C will receive the intervention supplement `Afzaaish’ soon after enrollment. 
Each enrolled LW in this arm will receive a routine supply of Afzaaish sachets (daily or weekly, depending 
on follow-up schedule). Sachet will be provided by trained research staff, either at the clinic or at the LW’s 
home depending on nature of follow-up visit. LWs in this arm will be counseled to consume two sachets 
in a 24-hour period (2 sachet per day). The sachets will be provided in routine installments until the child 
reaches the age of 6 months. On every visit, the LW will return the empty sachets and in exchange will 
receive new ones.  
Azithromycin 
Azithromycin will be given to infants at 42 days of age of LW randomized in Arm C and administered orally 
as a single dose using 20mg/kg as a standard for dosing. The suspension will be reconstituted by the 
trained staff and the dose will be calculated according to the infant’s weight, before administering to the 
subject. For dose accuracy 1 bottle of Azithromycin will be reconstituted and used for each infant, while 
the leftover dose will be archived for future reference purpose.  

 
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Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
6.10. 
Assessment and Recording Data on Study Participants  
The number of times each participant will be contacted for outcome assessment is summarized below 
(Table 4): 
1. Maternal and infant anthropometry at birth (baseline, within 0-6 days of birth) and then on 
monthly basis until 6 months of age. 
2. Every month, 24 hours food recall data will be collected from  every enrolled LW 
 

 
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Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
6.11. 
Laboratory sampling for study purpose only  
a) Breast milk samples on 50 randomly selected women from each arm will be collected for future 
assessment of micronutrients, macronutrients and microbiome. Aseptic hand expression technique 
will be followed for collection of breast milk for microbiome analysis (for Biobank for further analysis 
in future) 
b) Stool specimens will be collected from the same 50 randomly selected LWs and their infants from 
each arm for TaqMan Array Card (TAC) and Myeloperoxidase (MPO) using metagenomics approach 
(for Biobank for further analysis in future) 
c) Blood specimen will also be collected from the same 50 randomly selected LWs for the purpose of 
checking  hemoglobin levels, serum ferritin, transferrin receptor and acute phase proteins such as 
AGP and CRP and for Biobank for further analysis in future for proteomics. 
d) Infant anemia, hemoglobin, serum ferritin, transferrin receptor and acute phase proteins such as 
AGP and CRP and for Biobank for further analysis in future for proteomics (for all enrolled subjects 
upon consent from parents). 
6.12. 
Follow ups 
There will be specific assessment teams involved in this trial who will conduct their specific follow ups as 
per the below mentioned schedule to assess the newborn periodically and also measure the compliance 
of Afzaaish (Arm B and C) also providing routine counseling to all participants (all arms). Furthermore, the 
follow-up team will comprise of research staff that will be responsible for monthly 24 hour food recall 
checks. Additionally, they will also be responsible for assessing exclusive breastfeeding status, i.e. 
exclusive, predominant or partially, in all arms.  24 hours exclusive breastfeeding recall related data will 
be collected on the visit days. Child illness related history, care seeking, hospitalization and medication 
related history will be recorded at each visit. These follow up teams will also be in charge of household 
health promotion and education counseling for all the arms following the already established standard 
care counseling procedures. Exclusive breastfeeding will be assiduously promoted and precautionary 
counseling will be done regarding risks associated with giving the infant solids or liquids other than breast 
milk. 
Also an outcome assessment team (Anthropometry team) will be responsible to follow babies on a 
monthly basis until the age of 6 months to perform anthropometry on both LW and infants.  Schedule for 
follow-ups are as follows: 
 
 

 
Page 23 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
Activity 
Follow-up days, according to the age of the child 
after enrollment 
1. Afzaaish Compliance, newborn assessment 
(WHO algorithm of danger sign), past illness 
history, hospitalization related history, other 
care seeking, medication related history, 24 
hour EBF recall, Afzaaish compliance  
1-44 days are meant for pre and post Azithromycin 
dose) 
1. 1 
2. 2 
3. 3 
4. 4 
5. 5 
6. 6 
7. 7 
8. 8 
9. 9 
10. 10 
11. 11 
12. 12 
13. 13  
14. 15  
15. 17  
16. 19  
17. 21 
18. 23 
19. 25 
20. 29 
21. 32 
22. 35 
23. 38 
24. 41 
25. 42 
26. 43 
27. 44 
28. 48 
29. 52 
30. 59 
31. 67 
32. 74 
33. 81 
34. 88 
35. 95 
36. 102 
37. 109 
38. 116 
39. 123 
40. 130 
41. 137 
42. 144 
43. 151 
44. 158 
45. 165 
46. 172 
47. 179 
 
2. 24 hours food recall 
1. 7 
2. 35 
3. 67 
4. 95 
5. 123 
6. 151 
3. Anthropometry and vaccination record 
4. 27 
5. 56 
6. 85 
7. 114 
8. 143 
9. 177 
 
 
 
 
 
 
 
 

 
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7. OUTCOME ASSESSMENT 
7.1. 
Primary Outcome 
a. Length  velocity at 6 months of infant’s age 
Mean difference in length velocity of >0.12cm/month to look at comparisons between 
multiple arms 
7.2. 
Secondary Outcomes  
a. Growth velocity at 6 months of infant’s age 
Mean difference in growth velocity of >0.4 gram/kg/day to look at comparisons between 
multiple arms 
b. Length-for-Age Z-score (LAZ) at 6 months of infant’s age 
Mean difference in LAZM >0.5 to look at comparisons between multiple arms 
c. Weight-for-Length Z-score (WLZ) at 6 months of infant’s age 
Mean difference in WLZ >0.5 to look at comparisons between multiple arms 
d. Weight-for-Age Z-score of (WAZ) at 6 months of infant’s age 
Mean difference in WAZM >0.5 to look at comparisons between multiple arms 
Other Pre-specified Outcomes 
a. Breast milk composition of 50 randomly selected LWs 
To assess the quality of breast milk (macro and micronutrients) and microbiome analysis 
b. Stool specimens of 50 randomly selected LWs and Infants 
Stool will be collected for TaqMan Array Card (TAC) analysis of enteric pathogens and 
Myeloperoxidase (MPO) as a marker of gut inflammation and will be assessed using a 
metagenomics approach. 
c. Blood Specimens of 50 randomly selected LWs and all Infants enrolled in the trial 
To assess hemoglobin, serum ferritin, transferrin receptor and acute phase proteins such as AGP 
and CRP.  
 
 
 
 
 

 
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Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
7.3. 
Trial procedures for outcome assessment  
 
Assessments/ 
Specimen 
Arm 
At 
enrolment 
On each visit 
till age of 6 
months 
Monthly 
till age of 6 
months 
At 
40-42 
day 
of 
infants age  
(Before 
AZM dose) 
At 56 day 
of infants 
age 
At the age of 6 
months (study 
endpoint) 
Assessments at field 
Afzaaish 
Sachet 
distribution 
and 
Compliance 
B and C 
✓ 
✓ 
 
 
 
 
Azithromycin  
C 
 
 
 
✓ 
At 42 days 
 
 
Vital status 
A, B, C 
✓ 
✓ 
 
 
 
 
Standard nutrition 
and 
health 
promotion 
counseling  
A, B, C 
✓ 
✓ 
 
 
 
 
Infant 
anthropometry  
A, B, C 
✓ 
 
✓ 
 
 
✓ 
Maternal 
anthropometry 
A, B, C 
✓ 
 
✓ 
 
 
✓ 
24 
hours 
Food 
Recall 
A, B, C 
✓ 
 
✓ 
 
 
✓ 
Lab investigations for study purpose  (all Infants, where family is agree) 
Hemoglobin 
A, B, C 
 
 
 
✓ 
✓ 
 
Serum Ferritin 
A, B, C 
 
 
 
✓ 
✓ 
 
Transferrin 
receptors 
A, B, C 
 
 
 
✓ 
✓ 
 
AGP 
A, B, C 
 
 
 
✓ 
✓ 
 
CRP 
A, B, C 
 
 
 
✓ 
✓ 
 
Proteomics 
(Biobank) 
 
 
 
 
✓ 
✓ 
 
Stool specimens 
(50 infants only) 
A, B, C 
 
 
 
✓ 
✓ 
 
Lab investigations for Bio bank purpose only (50 LWs only) 
Breast milk 
 
A, B, C 
 
 
 
✓ 
✓ 
 
Stool specimens 
 
A, B, C 
 
 
 
✓ 
✓ 
 
Hemoglobin 
A, B, C 
 
 
 
✓ 
✓ 
 
Serum Ferritin 
A, B, C 
 
 
 
✓ 
✓ 
 
Transferrin 
receptors 
A, B, C 
 
 
 
✓ 
✓ 
 
AGP 
A, B, C 
 
 
 
✓ 
✓ 
 
CRP 
A, B, C 
 
 
 
✓ 
✓ 
 
Proteomics 
(Biobank) 
A, B, C 
 
 
 
✓ 
✓ 
 
 
 
 
 

 
Page 26 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
8. ADVERSE OUTCOMES 
8.1. 
Afzaaish supplement related – lactating woman only 
The trial participants will be instructed/counseled to report any adverse events (minor or major), which 
may be serious or non-serious in nature. The reporting of the adverse event will take into account any 
event which may be due to the use of nutritional supplement or with the use of any adjunct therapy, or 
even death of the trial registered participant (due to any circumstance). Nutravigilance is the term used 
for assessment, understanding, reporting, and prevention of any kind of adverse effects related to the use 
of food or nutritional supplement.  The reporting system for adverse event for dietary and nutritional 
supplement will follow the Food and Drug Administration (FDA) guidelines so that the information 
gathered from LW can be used for generating signals/alerts for possible public health concerns and 
subsequently assessing those signals and taking appropriate safety actions since this nutritional 
supplement used by LW will ultimately be associated with the health and outcomes of the infants.  
Reporting of adverse events on nutritional and dietary supplement includes the range of events from 
nausea, vomiting, diarrhea, abdominal distension, abdominal pain and rash. LW will be counseled to 
immediately report any serious condition which appears during the enrollment period, either during 
follow-up visits by research team or any time through a call on the number provided to them. For this trial 
the data collected on adverse events and other related reports will be provided by Principal Investigator 
directly to the DSMB on a monthly basis (serious) in the form of a report, while any fatal adverse events 
will be reported to DSMB and AKU/VPT IRB within 72 hours of event reported. On every follow-up visit, 
the research team will collect information for any potential side-effect and health status of LW through 
active and vigilant surveillance.  
Adverse and serious adverse event reporting and categorization 
The following adverse events will be observed for and documented: 
1. Vomiting: The forceful expulsion of gastric contents reported by LW. 
2. Diarrhea: Diarrhea is defined as the passage of three or more loose or liquid stools per day (or 
more frequent passage than is normal for the LW), reported by LW. 
3. Abdominal distention or pain: Conditions/symptoms reported by LW as feeling full, tight, or 
swollen in the abdomen, i.e. abdomen reported as swollen (distended), hard, and painful, 
reported by LW. 
4. Rash: An appearance of itchy, red, painful, and irritated skin, visible to health worker or reported 
by LW.  

 
Page 27 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
5. Any other serious symptom: If LW has reported any unusual symptoms, which she has never 
experienced before. 
Documentation of adverse event 
Adverse event reporting form will be filled against every event which is reported during the course of the 
study period and in case of any death the routine verbal autopsy form will also be filled. Based on the 
information provided in the forms, the decision will be made on whether a serious or non-serious adverse 
event is related to trial intervention. 
Serious Adverse Event 
Any adverse event will be labeled as a “Serious Adverse Event” (SAE) if it leads to: 
a. Intravenous therapy 
b. Hospitalization 
c. Life threatening situation  
d. Death 
Narrative description of serious adverse event will be provided in a document template and attached into 
the participants file. After reporting of any suspected adverse event, the senior research team/leader, 
including the study physicians will define the linkages of event with Afzaaish and fill an adverse event 
reporting form which will define the event in relation to Afzaaish as: 
 
a) Not related  
b) Unlikely related 
c) Possibly related 
d) Definitely related 
e) Not assessable 
 
 
 
 
 
 
 
 

 
Page 28 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
8.2. 
Azithromycin related – Infant  only 
To establish safety profile for AZM prophylaxis dose, the infant will be monitored for the next 48 hours. 
After administration, data will be collected and any side-effects (observed or reported) will be noted by 
the research team. They will revisit the infants after 24 hours (i.e. after dose, two consecutive visits will 
be performed with a 24 hour gap). For this trial the data collected on adverse events and other related 
reports will be provided by Principal Investigator directly to the DSMB on a monthly basis (serious) in the 
form of report, while any fatal adverse events will be reported to DSMB and AKU/VPT IRB within 72 hours 
of event reported.  
Adverse event reporting form will be filled against every event which is reported over the course of study 
period and in case of any death the routine verbal autopsy form will also be filled. Based on the 
information provided in the forms, decision will be made on whether to classify the event as serious or 
non-serious and also if it was related to trial intervention or not. Post-dose (43 and 44 day) visits will be 
performed to monitor and observe any suspected adverse event in the infants which are most likely to 
appear after AZM administration. If in any case, the dose is delayed, the 2 consecutive visits days will be 
changed accordingly. 
Adverse and serious adverse event reporting and categorization 
Based on the literature (30), following potential adverse event in infants related to AZM will be monitored: 
i. 
Gastrointestinal:  
a. Vomiting: Persistent vomiting or projectile vomiting in an infant reported by 
mother, within 24 hours of dose administration (Observed or reported).  
b. Poor feeding/suck: If an infant develops poor feeding/suck or is unable to feed 
within 24 hours of dose administration (Observed or reported). 
c. Necrotizing Enterocolitis: Abdominal distention/tenderness, bowel movements, 
signs of infection such as apnea (stop breathing), and lethargy within 24 hours of 
dose administration in an infant (Observed or reported). 
d. Diarrhea: Loose stool in an infant reported by mother, lead to severe dehydration, 
within 24 hours of dose administration (Observed or reported). 
ii. 
Respiratory:  
a. Respiratory distress: Fast breathing (60 or more breathes per minutes) and/or 
severe chest in drawing and/or baby turn bluish, within 24 hours of dose 
administration (Observed or reported). 

 
Page 29 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
iii. 
Rash: An appearance of itchy, red, painful, and irritated skin, visible by health worker or 
reported by family, within 24 hours of dose administration. 
Documentation of adverse event 
Adverse event reporting form will be filled against every event which is reported during the course of the 
study period and in case of any death the routine verbal autopsy form will also be filled. Based on the 
information provided in the forms, the decision will be made on whether a serious or non-serious adverse 
event is related to trial intervention. 
Serious Adverse Event 
Any adverse event will be labeled as a “Serious Adverse Event” (SAE) if it leads to: 
a. Intravenous therapy 
b. Hospitalization 
c. Life threatening situation  
d. Death 
Narrative description of serious adverse event will be provided in a document template and attached in 
the participants file. After reporting of any suspected adverse event, the senior research team/leader, 
including the study physicians will define the linkages of event with AZM and fill an adverse event 
reporting form which will define the event in relation to AZM as: 
a) Not related  
b) Unlikely related 
c) Possibly related 
d) Definitely related 
e) Not assessable 
 
 
 
 
 
 

 
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9. COMPLIANCE WITH AFZAAISH SUPPLEMENT AND AZITHROMYCIN 
Although, every woman enrolled and randomized, will be included in intention to treat analysis, however 
for the purpose of trial administration and assurance of maximum compliance, the following criteria will 
be followed to define compliance: 
a) Team will be tasked to ensure at least 75%  compliance in each month  with the consumption of  
a supplement which will be given to LW enrolled in Arm B and C 
 
Compliance 
(for 6 months) 
100% Compliance in each 
month 
75% Compliance in each 
month 
Low compliance and 
concern for counseling 
Afzaaish related- 
2 sachets of Afzaaish to 
be consumed in 24 
hours 
If 60-65 sachets  
consumed in 30 days 
If 45-49 sachets 
consumed in 30 days 
LW who has consumed 
<45 sachets for 2 
consecutive months 
(based on each 
month’s compliance) 
 
b) Team will ensure that dose of Azithromycin was administered on 42 days (window period of 7 
days). 
c) Exclusive Breastfeeding: Team will be tasked to achieve  at least 75% compliance  each month in 
all  arms  
Compliance 
(for 6 months) 
100% Compliance in each 
month 
75% Compliance in each 
month 
Low compliance and 
concern for counseling 
Only breastfeeding 
from LW enrolled in 
the trial, with no other 
things to drink or eat 
given to child 
Only breastfeeding 
from LW enrolled in 
the trial, with no other 
things to drink or eat 
given to child, 
confirmed on each visit 
If based on 24 hours 
recall, each month 
cumulative % of breast 
milk attempt is within 
75% of  the acceptable 
limit 
If based on  24 hours 
recall, each month 
cumulative % of breast 
milk attempt  
calculated is <75% ,it 
falls under the non per 
protocol limit 
 
 
 
 
 
 

 
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10. STATISTICAL ANALYSIS 
General approach to data analysis 
1. For the main study outcomes length velocity of the infant will be analyzed by predefined 
hypothesis in multiple arms. However conclusions on this part of the study will be based on formal 
hypothesis testing. 
2. For secondary objectives (weight velocity, length-for-age, weight-for-length, and weight-for-age 
Z scores) will be analyzed by predefined hypothesis in multiple arms. However conclusions on this 
part of the study will be based on formal hypothesis testing. 
Hypotheses to be tested 
Primary 
a) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product to lactating women of Mid-upper-arm-circumference (MUAC) of less than 
23.0 cm for initial 6 month of exclusive breastfeeding with or without single prophylaxis dose of 
Azithromycin to infants at day 42 of age, in improving length velocity as the primary outcome at 
6 months of age as compared to the standard of care.. 
Hypothesis to be tested: 
Ha = Mean difference in length velocity of >0.12cm/month to look at comparisons between 
multiple arms 
Ho = Mean difference in length velocity of ≤ 0.12cm/month to look at comparisons between 
multiple arms 
Secondary objective 
a) To assess the efficacy of a fortified, balanced energy-protein supplementation intake using a 
ready-to-use product to lactating women of Mid-upper-arm-circumference (MUAC) of less than 
23.0 cm for initial 6 month of exclusive breastfeeding with or without single prophylaxis dose of 
Azithromycin to infants at day 42 of age, on weight velocity, length-for-age, weight-for-length, 
and weight-for-age Z scores as secondary outcome at 6 months of age as compared to the 
standard of care. 
Hypothesis to be tested: 
i. 
Growth velocity at 6 months of infant’s age 
Ha = Mean difference in growth velocity of >0.4 gram/kg/day to look at comparisons between 
multiple arms 

 
Page 32 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
Ho = Mean difference in growth velocity of ≤ 0.4 gram/kg/day to look at comparisons between 
multiple arms 
ii. 
Length-for-Age Z-score (LAZ) at 6 months of infant’s age 
Ha = Mean difference in LAZ >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in LAZ ≤0.5 to look at comparisons between multiple arms 
iii. 
Weight-for-Length Z-score (WLZ) at 6 months of infant’s age 
Ha = Mean difference in WLZ >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in WAZ ≤0.5 to look at comparisons between multiple arms 
iv. 
Weight-for-Age Z-score of (WAZ) at 6 months of infant’s age 
Ha = Mean difference in WAZ >0.5 to look at comparisons between multiple arms 
Ho = Mean difference in WAZ ≤0.5 to look at comparisons between multiple arms 
 
Data cleaning and procedures 
Data cleaning process will be done by these procedures: 
1. Data analyst will clean the required data for the main analyses. At this point, all      
investigators will be blinded to the intervention each participant has been receiving. 
2. The study statisticians will review the data and complete preliminary analyses for group 
comparisons (without knowing the actual interventions). 
 
Definition of key variables 
i. 
Length velocity 
Length velocity to be calculated from baseline length measurement of the infants taken within 
0-6 days of life and subsequent measurement in ‘cm’ taken at each 4 weeks interval, until the 
age of 6 month, calculated as cm/month.  
ii. 
Growth velocity 
Growth velocity to be calculated from baseline weight measurement of the infants taken 
within 0-6 days of life and subsequent measurement in ‘gram’ taken at each 4 weeks interval, 
until the age of 6 month, calculated as gm/kg/month.  
iii. 
Length-for-Age Z-score (LAZ) at 6 months of infant’s age 
Length-for-age will be calculated from age, sex, and length information from the baseline 
measurements within 0-6 days of birth and infant anthropometry on monthly basis until 6 

 
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months of age by using the STATA WHO packages using the WHO 2006 multi-centre growth 
standard. The values will be expressed as Z-score units, with two decimals.  
iv. 
Weight-for-Length Z-score (WLZ) at 6 months of infant’s age  
Weight-for-length will be calculated from sex, weight and length information from the 
baseline measurements within 0-6 days of birth and infant anthropometry on monthly basis 
until 6 months of age by using the STATA WHO packages using the WHO 2006 multi-centre 
growth standard. The values will be expressed as Z-score units, with two decimals.  
v. 
Weight-for-Age Z-score of (WAZ) at 6 months of infant’s age 
Weight-for-Age will be calculated from weight, sex and gender information from the baseline 
measurements within 0-6 days of birth and infant anthropometry on monthly basis until 6 
months of age by using the STATA WHO packages using the WHO 2006 multi-centre growth 
standard. The values will be expressed as Z-score units, with two decimals.  
 
Analysis approach 
Statistical analysis will be performed by Stata, version 15. The baseline characteristics will be 
assessed by arm. The primary analysis will be an intention-to-treat (ITT) analysis. We will compare 
the mean length velocity (cm/ month) (primary outcome), weight velocity (gm/kg/day), LAZ, WLZ, 
and WAZ between the two intervention arms and the control arm using  ANOVA, with the model 
adjusted for the birth weight and age of the infants at enrollment. If an outcome is missing for the 
intention-to-treat infants, the means from that group will be imputed.  
Safety outcomes 
i. 
Maternal serious adverse events 
The occurrence of maternal SAEs will be expressed as the proportion of women with at least 
one SAE during the follow-up period (from enrolment to 179th day). The proportion will be 
calculated by dividing the number of women with at least one recorded SAE by the total 
number of enrolled participants. Results will be shown both as proportions of participants 
with any SAE as well as tabulated by the SAE category (death, hospitalization, other). If any 
participant has experienced more than one type of SAE, the participant will be recorded in 
each category.  
ii. 
Infant serious adverse events 
The occurrence of infant SAEs will be expressed as the proportion of babies with at least one 
SAE during the follow-up period (from enrolment to 179th day). The proportion will be 

 
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calculated by dividing the number of babies with at least one recorded SAE by the total 
number of recorded newborns. Results will be shown both as proportions of participants with 
any SAE as well as tabulated by the SAE category (death, hospitalization, other). If any 
participant has experienced more than one type of SAE, the participant will be recorded in 
each category.  
 
Interim analysis 
Data Safety and Monitoring Board of the trial will request an independent statistician to run interim 
analysis when 50% of the enrollment will complete the 6 months follow-ups in the trial. The analysis will 
be coded and the findings will on difference in primary, secondary outcomes and Serious Adverse Events 
will be shared to DSMB in blinded coding. The DSMB will review results from the interim data analysis and 
will determine whether stopping boundaries have been crossed. Additional interim analyses may be 
completed and reviewed by the DSMB, per the advisement of the DSMB chair. The DSMB may, at its own 
discretion, ask the interim analysis to be done earlier or additional interim analyses to be completed on 
an ad hoc basis. 
For any interim analysis, the data management team at the VPT will prepare a blinded, cleaned analysis 
database. The interim analysis will be conducted on a blinded basis, as follows: Project data team at VPT 
will prepare and verify interim analysis programs using randomly generated dummy treatment 
assignments that have no relationship to the true intervention assignments. An independent statistician 
will subsequently incorporate blinded treatment codes group 1, 2 and 3 for safety end points status. The 
data team will separately provide treatment codes to the chair of the DSMB in a sealed file. At a minimum, 
the results to be presented will include a summary of screening and enrollment status, descriptive 
statistics of baseline variables, summaries of protocol violations, serious adverse events, primary, and 
secondary growth outcome. Site-specific and overall results will be pooled over treatment groups. DSMB 
may request other analyses as it deems fit. The DSMB will decide whether or not they wish to open the 
treatment code envelopes to un-blind themselves.  
i. 
Stopping guidelines / Stopping rules 
Based on the trial safety monitoring and interim analysis, the following may be the key 
decision/s of DSMB pertaining to the trial:  
a) No action needed, trial continues as planned. 
b) Early stopping due, for example, to clear benefit or harm of an intervention/s, 
uselessness, or external evidence. 

 
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c) Extending recruitment (based on actual control arm response rates being different to 
predicted rather than on emerging differences) or extending follow-up. 
d) Stopping an arm of the multi-arm trial. 
e) Sanctioning and/or proposing protocol changes 
While stopping rules will be utilized to help the DSMB to assess the justification of trial continuation, the 
DSMB will take into account the balance of risks and benefits as well as consistency with external 
evidence. Thus, the DSMB recommendation to continue or discontinue will not be based on any single 
issue or value in any of the analyses, but a comprehensive analysis taking into account multiple issues 
around the trial. 
The DSMB may recommend termination or modification of the study if: 
i. 
In an interim analysis, there is strong evidence of a benefit (increases length velocity 
and/or secondary outcome) from the study intervention (O’Brien Fleming rule, p<0.005). 
ii. 
In an interim analysis, there is strong evidence of harm (incidence of SAEs) from the study 
intervention. For harm assessment, no fixed statistical rules will be applied, but the DSMB 
will holistically consider point estimates and confidence intervals for SAE incidence 
differences, p-values from appropriate statistical tests and other relevant factors, when 
determining its recommendation about study continuation or discontinuation.  
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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11. SAMPLE SIZE CALCULATIONS & POWER ANALYSIS 
The sample size is calculated and powered to look at multiple comparisons, between arms A and B, A and 
C plus B and C with a hypothesized higher increase with addition of Azithromycin to infant with Afzaaish 
in LW. Also, adjustment for multiple comparison is needed. Thus, calculations for the sample size is 
estimated and are per arm sample size is provided below based on the primary outcome of length velocity 
in LW trial with 1-sided testing, alphas to account for multiple comparison (the lower alpha), and drop out 
of 14%  is assumed in the trial.  
Assumptions for Sample Size Calculations 
Variable 
Differenc
e 
Std 
Dev 
Alpha 
Drop 
out 
portion 
n/arm 
n/arm 
adj 
dropout 
Actual Power 
Length Velocity 
0.12 
0.5 
0.025 
0.14 
274 
319 
0.801 
 
Based on assumptions, a sample size of 319 children per arm is calculated, assuming 14% drop out rate 
(i.e.  10% margin for loss to follow up, and 4% infants deaths).   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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12. REFRENCES 
1. 
Arunda MO, Agardh A, Asamoah BO. Survival of low birthweight neonates in Uganda: analysis of 
progress between 1995 and 2011. BMC pregnancy and childbirth. 2018; 18(1):189. 
2. 
Morrison JL, Regnault TR. Nutrition in pregnancy: optimising maternal diet and fetal adaptations 
to altered nutrient supply. Multidisciplinary Digital Publishing Institute; 2016. 
3. 
Lee AC, Kozuki N, Cousens S, Stevens GA, Blencowe H, Silveira MF, et al. Estimates of burden and 
consequences of infants born small for gestational age in low and middle income countries with 
INTERGROWTH-21st standard: analysis of CHERG datasets. bmj. 2017; 358:j3677. 
4. 
Kozuki N, Katz J, Lee AC, Vogel JP, Silveira MF, Sania A, et al. Short maternal stature increases risk 
of small-for-gestational-age and preterm births in low-and middle-income countries: Individual 
participant data meta-analysis and population attributable fraction. 2015. 
5. 
Christian P, Mullany LC, Hurley KM, Katz J, Black RE, editors. Nutrition and maternal, neonatal, 
and child health. Seminars in perinatology; 2015: Elsevier. 
6. 
Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, De Onis M, et al. Maternal and child 
undernutrition and overweight in low-income and middle-income countries. The lancet. 2013; 
382(9890):427-51. 
7. 
Organization WH. The double burden of malnutrition: policy brief. 2016. 
8. 
Bhutta Z, Soofi S, Zaidi S, Habib M, Hussain I. National nutrition survey Pakistan. Karachi, Pakistan: 
Aga Khan University, Pakistan. 2011. 
9. 
Amza A, Sun NY, Kadri B, Nassirou B, Stoller NE, Zhou Z, et al. Does mass azithromycin distribution 
impact child growth and nutrition in Niger? A cluster-randomized trial. PLoS neglected tropical diseases. 
2014; 8(9):e3128. 
10. 
Keenan JD, Bailey RL, West SK, Arzika AM, Hart J, Weaver J, et al. Azithromycin to Reduce 
Childhood Mortality in Sub-Saharan Africa. New England Journal of Medicine. 2018; 378(17):1583-92. 
11. 
Porco TC, Gebre T, Ayele B, House J, Keenan J, Zhou Z, et al. Effect of mass distribution of 
azithromycin for trachoma control on overall mortality in Ethiopian children: a randomized trial. Jama. 
2009;302(9):962-8. 
12. 
Keenan JD, Ayele B, Gebre T, Zerihun M, Zhou Z, House JI, et al. Childhood mortality in a cohort 
treated with mass azithromycin for trachoma. Clinical infectious diseases. 2011; 52(7):883-8. 
13. 
Adu-Afarwuah S, Lartey A, Okronipa H, Ashorn P, Peerson JM, Arimond M, et al. Small-quantity, 
lipid-based nutrient supplements provided to women during pregnancy and 6 mo postpartum and to their 
infants from 6 mo of age increase the mean attained length of 18-mo-old children in semi-urban Ghana: 
a randomized controlled trial, 2. The American journal of clinical nutrition. 2016; 104(3):797-808. 
14. 
Ashorn P, Alho L, Ashorn U, Cheung YB, Dewey KG, Gondwe A, et al. Supplementation of Maternal 
Diets during Pregnancy and for 6 Months Postpartum and Infant Diets Thereafter with Small-Quantity 
Lipid-Based Nutrient Supplements Does Not Promote Child Growth by 18 Months of Age in Rural Malawi: 
A Randomized Controlled Trial–4. The Journal of nutrition. 2015;145(6):1345-53. 
15. 
Lanou H, Huybregts L, Roberfroid D, Nikièma L, Kouanda S, Van Camp J, et al. Prenatal nutrient 
supplementation and postnatal growth in a developing nation: an RCT. Pediatrics. 2014:peds. 2013-850. 
16. 
Imdad A, Bhutta ZA. Nutritional management of the low birth weight/preterm infant in 
community settings: a perspective from the developing world. The Journal of pediatrics. 
2013;162(3):S107-S14. 
17. 
Ota E, Hori H, Mori R, Tobe-Gai R, Farrar D. Antenatal dietary education and supplementation to 
increase energy and protein intake. Cochrane Database of Systematic Reviews. 2015(6). 
18. 
Haider BA, Bhutta ZA. Multiple-micronutrient supplementation for women during pregnancy. The 
Cochrane Library. 2017. 

 
Page 38 of 38 
Version 1.3 January 16, 2019 (Updated for Plan on analysis) 
 
19. 
Adu-Afarwuah S, Lartey A, Brown KH, Zlotkin S, Briend A, Dewey KG. Randomized comparison of 
3 types of micronutrient supplements for home fortification of complementary foods in Ghana: effects on 
growth and motor development–. The American journal of clinical nutrition. 2007;86(2):412-20. 
20. 
Phuka JC, Maleta K, Thakwalakwa C, Cheung YB, Briend A, Manary MJ, et al. Complementary 
feeding with fortified spread and incidence of severe stunting in 6-to 18-month-old rural Malawians. 
Archives of pediatrics & adolescent medicine. 2008;162(7):619-26. 
21. 
Arimond M, Zeilani M, Jungjohann S, Brown KH, Ashorn P, Allen LH, et al. Considerations in 
developing lipid-based nutrient supplements for prevention of undernutrition: experience from the I 
nternational L ipid-B ased N utrient S upplements (iLiNS) P roject. Maternal & child nutrition. 2015;11:31-
61. 
22. 
Nair V, Loganathan P, Soraisham AS. Azithromycin and other macrolides for prevention of 
bronchopulmonary dysplasia: a systematic review and meta-analysis. Neonatology. 2014; 106(4):337-47. 
23. 
Smith C, Egunsola O, Choonara I, Kotecha S, Jacqz-Aigrain E, Sammons H. Use and safety of 
azithromycin in neonates: a systematic review. BMJ open. 2015; 5(12):e008194. 
24. 
Thinkhamrop J, Hofmeyr GJ, Adetoro O, Lumbiganon P, Ota E. Antibiotic prophylaxis during the 
second and third trimester to reduce adverse pregnancy outcomes and morbidity. Cochrane database of 
systematic reviews. 2015(1). 
25. 
Unger HW, Ome-Kaius M, Wangnapi RA, Umbers AJ, Hanieh S, Suen CSLW, et al. Sulphadoxine-
pyrimethamine plus azithromycin for the prevention of low birthweight in Papua New Guinea: a 
randomised controlled trial. BMC medicine. 2015; 13(1):9. 
26. 
Solomon AW, Mohammed Z, Massae PA, Shao JF, Foster A, Mabey DC, et al. Impact of mass 
distribution of azithromycin on the antibiotic susceptibilities of ocular Chlamydia trachomatis. 
Antimicrobial agents and chemotherapy. 2005; 49(11):4804-6. 
27. 
Parnham MJ, Haber VE, Giamarellos-Bourboulis EJ, Perletti G, Verleden GM, Vos R. Azithromycin: 
mechanisms of action and their relevance for clinical applications. Pharmacology & therapeutics. 2014; 
143(2):225-45. 
28. 
Giamarellos-Bourboulis EJ, Pechere J-C, Routsi C, Plachouras D, Kollias S, Raftogiannis M, et al. 
Effect of clarithromycin in patients with sepsis and ventilator-associated pneumonia. Clinical Infectious 
Diseases. 2008;46(8):1157-64. 
29. 
Doan T, Arzika AM, Ray KJ, Cotter SY, Kim J, Maliki R, et al. Gut microbial diversity in antibiotic-
naive children after systemic antibiotic exposure: a randomized controlled trial. Clinical Infectious 
Diseases. 2017; 64(9):1147-53. 
30. 
Eberly MD, Eide MB, Thompson JL, Nylund CM. Azithromycin in early infancy and pyloric stenosis. 
Pediatrics. 2015: peds. 2014-26.