Lactoferrin and Lysozyme Supplementation for Long-term Diarrhea Sequelae

Study Overview
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Brief Summary
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Children in low- and middle-income countries who are hospitalized for diarrhea and also have malnutrition are at high risk for illness and death in the 6 months period following treatment for diarrhoea despite receiving current guideline recommended diarrhea management (such as oral rehydration solution, or ORS). This study will test whether nutritional supplements made from milk (lactoferrin or lysozyme) or a combination of the two (lactoferrin and lysozyme) will prevent children from having repeated diarrhea episodes and help improve their nutrition by improving their stomach health or preventing new disease during this 6-month period. The study is taking place at 7 hospitals in Western Kenya. Six hundred participants will be enrolled if they provide informed consent to participate, are aged 6-24 months, were hospitalized with diarrhea and malnutrition and have been managed by the facility nutritionists and ready to return home. Participation in the study will entail providing information on the child's health history, collection of stool samples, blood, and potentially urine. The caregiver will be provided sachets of the investigational product to take home and mix daily with their child's porridge or other complimentary food, and asked to return to the clinic 4 times in the subsequent 6 months, and also consent to having a community health worker visit their home every two weeks for a follow up visit. The risks to the participant and their caregiver are minimal. The information gained in this study will help us create new treatments and develop new strategies to treat sick children to prevent death and illness.

Detailed Description
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Current diarrhea management strategies in low- and middle-income countries (oral rehydration solution, ReSoMal and zinc) focus primarily on the management of dehydration and micronutrient replacement and appear to have negligible impact in preventing future diarrheal episodes or improving nutritional outcomes. Lactoferrin and lysozyme are milk-derived nutritional supplements that may reduce the risk of diarrheal episodes and accelerate nutritional recovery by treating or preventing underlying enteric infections and/or improving enteric function. Children with moderate or severe wasting are at particularly high-risk of death, diarrhea recurrence, and nutritional deterioration following a diarrheal episode. This factorial, double-blind, placebo-controlled, randomized trial aims to determine the efficacy of lactoferrin and lysozyme supplementation in decreasing diarrhea incidence and improving nutritional recovery in children convalescing from diarrhea and wasting. We will explore whether these interventions improve outcomes by reducing enteric pathogens, improving enteric function and/or increasing hemoglobin concentrations in these children. This study aims to enroll 600 Kenyan children aged 6-24 months from facilities in Western Kenya. Enrolled children will be randomized to 16-weeks of lactoferrin, lysozyme, a combination of the two, or placebo and be followed up for 24 weeks total, with bi-weekly home visits by community health workers and clinic visits at 4, 10, 16, and 24 weeks. Results of this study will inform management strategies for children with moderate/severe wasting at high risk for mortality, morbidity, and nutritional deterioration following diarrhea. Aim 1: To determine whether a 16-week course of lactoferrin, lysozyme or a combination of both shortens time to WHO-defined recovery from wasting (MUAC ≥12.5cm) and reduces the incidence of moderate-to-severe diarrhea during the subsequent 6-months following presentation to a health facility with diarrhea among children with moderate/severe childhood wasting (MUAC <12.5 cm at the time of screening). Hypothesis: Children randomized to lactoferrin, lysozyme, or the combination of both will experience a lower incidence of moderate-to-severe diarrhea and an earlier recovery from wasting (increased MUAC) over the subsequent 6-months than placebo-treated children. Combination therapy will provide synergistic benefit in reducing diarrhea and improving nutritional recovery. Aim 2: To explore whether a 16-week course of lactoferrin, lysozyme or combination therapy improves secondary clinical, nutritional, enteric pathogen, and enteric function outcomes. Hypothesis: Children randomized to lactoferrin, lysozyme, or the combination will experience fewer hospitalizations and deaths, improved linear growth, a reduced prevalence of specific enteric bacteria associated with linear growth failure (Campylobacter, LT-ETEC, EAEC, typical EPEC and/or Shigella), improved markers of enteric dysfunction (myeloperoxidase, alpha antitrypsin, neopterin, and the lactulose:rhamnose ratio) and improved hemoglobin, as compared to placebo-treated children. Aim 3: To evaluate acceptability, adherence and cost-effectiveness of lactoferrin and/or lysozyme in Kenya. Hypothesis: Both therapies will be highly acceptable to caregivers and health workers. Adherence to the therapies will be high among participants (≥ 95%). Lactoferrin and lysozyme, alone and in combination, will be more cost-effective interventions for reducing moderate-to-severe diarrhea in the short-term as compared to the standard-of-care.

Official Title
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Lactoferrin and Lysozyme Supplementation for Long-term Diarrhea Sequelae

Conditions
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Wasting, Diarrhea, Malnutrition, Child

Intervention / Treatment
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* Dietary Supplement: Lactoferrin
* Dietary Supplement: Lysosyme
* Combination Product: Lactoferrin + Lysosyme
* Other: Placebo


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Age 6-24 months Managed as an outpatient or inpatient for diarrhea at one of the recruiting sites MUAC <12.5 cm at the time of screening Plan to stay within the study area for the next 6 months or greater Exclusion Criteria: Age younger than 6 months or older than 24 months Caregiver does not provide consent to study participation History of 2 or more blood transfusions in the past 12 months Exclusively breastfeeding at the time of enrollment History of congenital defect or syndrome that prevents age-appropriate feeding (e.g. cleft palate) History of allergy to dairy products Child is not ready to return home (is not yet discharged), or discharged against medical advice Unwilling to participate in the dual sugar permeability sub-study if selected Enrollment in another study

Ages Eligible for Study
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Minimum Age: 6 Months
Maximum Age: 24 Months

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Factorial Assignment
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Lactoferrin Arm<br>16-week course of lactoferrin | Dietary Supplement: Lactoferrin<br>* Caregivers of children will be instructed to provide 41.5g of lactoferrin daily mixed with 125 mL of porridge or other complimentary food.<br>|
| Experimental: Lysosyme Arm<br>16-week course of lysozyome | Dietary Supplement: Lysosyme<br>* Caregivers of children will be instructed to provide 41.5g of lysosyme daily mixed with 125 mL of porridge or other complimentary food.<br>|
| Experimental: Combination Arm (Lactoferrin + Lysosyme)<br>16-week course of lactoferrin and lysosome | Combination Product: Lactoferrin + Lysosyme<br>* Caregivers of children will be instructed to provide 40 grams of Lysosure with 1.5 grams of bovine lactoferrin daily mixed with 125 mL of porridge or other complimentary food.<br>|
| Placebo Comparator: Placebo Arm<br>16-week course of taste/appearance-matched placebo | Other: Placebo<br>* Caregivers of children will be instructed to provide 41.5 grams of unmodified rice powder produced by Ventria Bioscience Inc daily mixed with 125 mL of porridge or other complimentary food.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Incidence of moderate-to-severe diarrhea | Defined as total number of new diarrhea episodes (> 48 hours after a diarrhea-free period) deemed moderate-to-severe, divided by the child-time at risk during the 6-month follow-up period. Moderate-to-severe diarrhea will be defined as ≥ 3 using the CODA (Community DiarrhoeA) diarrhea severity score or dysentery (evidence or reported visible blood in stool). | 6 months | 
| Time to nutritional recovery | Defined as the number of days since enrollment to the date of the second of two consecutive MUAC measurements ≥ 12.5 cm. | 6 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Incidence of diarrhea (any severity) | Diarrhea (any severity) will be defined as diarrhea (3 or more abnormally loose or watery stool) during follow-up, irrespective of severity, ascertained through follow-up visit questionnaires and a diarrhea diary. Episodes will be defined as per the primary outcome. | 6 months | 
| Incidence of severe diarrhea | Severe diarrhea defined by the CODA diarrhea severity score of 7 or more or dysentery. Episodes will be defined as per the primary outcome. | 6 months | 
| Incidence of dysentery | Dysentery will be defined as evidence or reported visible blood in stool. Episodes will be defined as per the primary outcome. | 6 months | 
| Incidence of medically-attended diarrhea | Medically-attended diarrhea will be defined as diarrhea that led to an outpatient or inpatient visit at a health facility or hospital that is typically attended by a nurse, clinical officer, and/or physician. Episodes will be defined as per the primary outcome. | 6 months | 
| Cumulative duration of diarrhea | Defined as cumulative days of diarrhea ascertained from follow-up visit questionnaires and a diarrhea diary, irrespective of episodes | 6 months | 
| Incidence of hospitalization | Defined as any inpatient admission that results in an overnight stay (irrespective of diagnosis) in a health-facility and time to hospitalization or death analyzed as a combined outcome | 6 months | 
| Composite outcome of time to hospitalization and death | Hospitalization will be defined as any inpatient admission that results in an overnight stay (irrespective of diagnosis) in a health-facility | 6 months | 
| Hemoglobin concentration | Will be determined at week 16 | Measured at week 16 | 
| Change in length for age z-score (linear growth) | Growth that includes length and weight measurements at each time point will be used to create age-standardized z-scores, calculated using WHO-established reference standards and the WHO ANTHRO software. Linear growth will be defined as change (Δ) in LAZ. | 6 months | 
| Change in weight for length z-score | Growth that includes length and weight measurements at each time point will be used to create age-standardized z-scores, calculated using WHO-established reference standards and the WHO ANTHRO software. Ponderal growth will be defined as change (Δ) in weight for length z-score (WLZ) and Δ MUAC. | 6 months | 
| Change in mid-upper arm circumference (cm) | Growth that includes length and weight measurements at each time point will be used to create age-standardized z-scores, calculated using WHO-established reference standards and the WHO ANTHRO software. Ponderal growth will be defined as change (Δ) in weight for length z-score (WLZ) and Δ MUAC. | 6 months | 
| Concentration of fecal alpha antitrypsin (mg/g) | Fecal biomarker of enteric function | Measured at baseline, week 4, 16, and 24 | 
| Concentration of fecal myeloperoxidase (ng/mL) | Fecal biomarker of enteric function | Measured at baseline, week 4, 16, and 24 | 
| Concentration of fecal calprotectin (mcg/g) | Fecal biomarker of enteric function | Measured at baseline, week 4, 16, and 24 | 
| Lactulose:rhamnose ratio | Lactulose:rhamnose ratio is a functional assessment of enteric integrity. Ratio will be measured in subset of 50 children per arm (200 children total). | Measured at week 4, 10, 16, and 24 for a subset of 200 participants (50 per arm) | 
| Proportion of participants with enteric infections | Determined by qPCR at or below the minimum limit of detection (cycle thresholds [CT] <35) | Measured at week 4 for all participants and at week 16 and 24 for a subset of 200 participants (50 per arm) | 
| Proportion of caregivers reporting that administration of lactoferrin and/or lysozyme was desirable or satisfactory via 5-point Likert scale responses in surveys and focus group discussions (FGDs) | Acceptability measure | Week 4, 10, 16 (surveys) and 6 months (FGDs) | 
| Proportion of caregivers reporting perceived trust, safety, and comfort in administering lactoferrin and/or lysozyme via 5-point Likert scale responses in surveys and focus group discussions (FGDs) | Acceptability measure | Week 16 (surveys) and 6 months (FGDs) | 
| Proportion of caregivers self-reporting their child consumed some or all of the investigational product ≥95% of the time using daily pictorial logs | Adherence measure | 16 weeks | 
| Proportion of children adherent to the recommended dosing based on objectively measured container consumption (± 10% consumption of prescribed IP in all weeks) | Adherence measure | 16 weeks | 
| Incremental costs of diarrhea | Incremental costs of diarrhea will be measured in each arm and compared to the placebo arm | 6 months | 
| Cost-per-episode of diarrhea averted | Cost-per-episode of diarrhea averted will be measured in each arm and compared to the placebo arm | 6 months |