Lactoferrin and Lysozyme Supplementation for Long-term Diarrhea Sequelae

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.

Study Overview

• Status: Recruiting
• Conditions: Diarrhea; Wasting; Malnutrition, Child
• Intervention / Treatment: Dietary supplement: Lactoferrin; Dietary supplement: Lysozyme; Combination product: Lactoferrin + Lysozyme; Other: Placebo

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 600
• Phase: Phase 3

Contacts and Locations

• Study Contact: Name: Ruchi Tiwari; Phone Number: 2067904389; Email: ruchit@uw.edu

Study Locations

• Kenya
  • Homa Bay, Kenya
    • Recruiting
    • Homa Bay County Referral Hospital
  • Isibania, Kenya
    • Recruiting
    • Isebania Sub-County Hospital
  • Kisii, Kenya
    • Recruiting
    • Kisii Teaching and Referral Hospital
  • Rongo, Kenya
    • Recruiting
    • Rongo Sub-County Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 6 months to 2 years (Child)
• Accepts Healthy Volunteers: Yes

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Factorial Assignment
• Masking: Quadruple

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Lactoferrin Arm; 16-week course of lactoferrin	Dietary supplement: Lactoferrin; Caregivers of children will be instructed to provide 1.5g of lactoferrin with 40g of unmodified rice powder daily mixed with 125 mL of porridge or other complimentary food.
Placebo Comparator: Placebo Arm; 16-week course of taste/appearance-matched placebo	Other: Placebo; Caregivers of children will be instructed to provide 41.5 grams of unmodified rice powder daily mixed with 125 mL of porridge or other complimentary food.
Experimental: Lysozyme Arm; 16-week course of lysozyme	Dietary supplement: Lysozyme; Caregivers of children will be instructed to provide 41.5g of Lysosure daily mixed with 125 mL of porridge or other complimentary food.
Experimental: Combination Arm (Lactoferrin + Lysozyme); 16-week course of lactoferrin and lysozyme	Combination product: Lactoferrin + Lysozyme; Caregivers of children will be instructed to provide 40g of Lysosure with 1.5 grams of lactoferrin daily mixed with 125 mL of porridge or other complimentary food.

What is the study measuring?

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
Proportion of children who recovered from wasting based on two consecutive MUAC measurements ≥12.5 at any point during the follow-up period.		6 months

Collaborators and Investigators

• Sponsor: University of Washington
• Collaborators: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)
• Investigators: Principal Investigator: Patricia B Pavlinac, University of Washington

Publications and helpful links

General Publications

• Tickell KD, Pavlinac PB, John-Stewart GC, Denno DM, Richardson BA, Naulikha JM, Kirera RK, Swierczewski BE, Singa BO, Walson JL. Impact of Childhood Nutritional Status on Pathogen Prevalence and Severity of Acute Diarrhea. Am J Trop Med Hyg. 2017 Nov;97(5):1337-1344. doi: 10.4269/ajtmh.17-0139.
• Talbert A, Ngari M, Bauni E, Mwangome M, Mturi N, Otiende M, Maitland K, Walson J, Berkley JA. Mortality after inpatient treatment for diarrhea in children: a cohort study. BMC Med. 2019 Jan 28;17(1):20. doi: 10.1186/s12916-019-1258-0.
• Tickell KD, Sharmin R, Deichsel EL, Lamberti LM, Walson JL, Faruque ASG, Pavlinac PB, Kotloff KL, Chisti MJ. The effect of acute malnutrition on enteric pathogens, moderate-to-severe diarrhoea, and associated mortality in the Global Enteric Multicenter Study cohort: a post-hoc analysis. Lancet Glob Health. 2020 Feb;8(2):e215-e224. doi: 10.1016/S2214-109X(19)30498-X.
• Brander RL, Pavlinac PB, Walson JL, John-Stewart GC, Weaver MR, Faruque ASG, Zaidi AKM, Sur D, Sow SO, Hossain MJ, Alonso PL, Breiman RF, Nasrin D, Nataro JP, Levine MM, Kotloff KL. Determinants of linear growth faltering among children with moderate-to-severe diarrhea in the Global Enteric Multicenter Study. BMC Med. 2019 Nov 25;17(1):214. doi: 10.1186/s12916-019-1441-3.
• Cheng WD, Wold KJ, Bollinger LB, Ordiz MI, Shulman RJ, Maleta KM, Manary MJ, Trehan I. Supplementation With Lactoferrin and Lysozyme Ameliorates Environmental Enteric Dysfunction: A Double-Blind, Randomized, Placebo-Controlled Trial. Am J Gastroenterol. 2019 Apr;114(4):671-678. doi: 10.14309/ajg.0000000000000170.
• Ochoa TJ, Chea-Woo E, Baiocchi N, Pecho I, Campos M, Prada A, Valdiviezo G, Lluque A, Lai D, Cleary TG. Randomized double-blind controlled trial of bovine lactoferrin for prevention of diarrhea in children. J Pediatr. 2013 Feb;162(2):349-56. doi: 10.1016/j.jpeds.2012.07.043. Epub 2012 Aug 30.
• Black RE, Victora CG, Walker SP, Bhutta ZA, Christian P, de Onis M, Ezzati M, Grantham-McGregor S, Katz J, Martorell R, Uauy R; Maternal and Child Nutrition Study Group. Maternal and child undernutrition and overweight in low-income and middle-income countries. Lancet. 2013 Aug 3;382(9890):427-451. doi: 10.1016/S0140-6736(13)60937-X. Epub 2013 Jun 6.
• Zavaleta N, Figueroa D, Rivera J, Sanchez J, Alfaro S, Lonnerdal B. Efficacy of rice-based oral rehydration solution containing recombinant human lactoferrin and lysozyme in Peruvian children with acute diarrhea. J Pediatr Gastroenterol Nutr. 2007 Feb;44(2):258-64. doi: 10.1097/MPG.0b013e31802c41b7.
• Tiwari R, Tickell KD, Yoshioka E, Otieno J, Shah A, Richardson BA, Keter L, Okello M, Nyabinda C, Trehan I, McGrath CJ, Means AR, Houpt ER, Liu J, Platts-Mills JA, Njunge JM, Rwigi D, Diakhate MM, Nyaoke J, Ochola E, John-Stewart G, Walson JL, Pavlinac PB, Singa BO. Lactoferrin and lysozyme to promote nutritional, clinical and enteric recovery: a protocol for a factorial, blinded, placebo-controlled randomised trial among children with diarrhoea and malnutrition (the Boresha Afya trial). BMJ Open. 2024 Aug 9;14(8):e079448. doi: 10.1136/bmjopen-2023-079448.

Study record dates

Study Major Dates

• Study Start (Actual): March 10, 2023
• Primary Completion (Estimated): July 1, 2026
• Study Completion (Estimated): December 1, 2026

Study Registration Dates

• First Submitted: August 3, 2022
• First Submitted That Met QC Criteria: August 24, 2022
• First Posted (Actual): August 29, 2022

Study Record Updates

• Last Update Posted (Estimated): September 11, 2025
• Last Update Submitted That Met QC Criteria: September 4, 2025
• Last Verified: August 1, 2025

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Nutrition Disorders; Metabolic Diseases; Signs and Symptoms, Digestive; Pathological Conditions, Signs and Symptoms; Nutritional and Metabolic Diseases; Signs and Symptoms; Child Nutrition Disorders; Diarrhea; Wasting Syndrome; Amino Acids, Peptides, and Proteins; Proteins; Substandard Drugs; Pharmaceutical Preparations; Carbohydrates; Glycoside Hydrolases; Hydrolases; Enzymes; Enzymes and Coenzymes; Globulins; Glycoproteins; Glycoconjugates; Endopeptidases; Peptide Hydrolases; Serine Endopeptidases; Serine Proteases; Dietary Proteins; Milk Proteins; Animal Proteins, Dietary; Carrier Proteins; Transferrins; Iron-Binding Proteins; Lactoglobulins; Whey Proteins; Metalloproteins; Lactoferrin; Muramidase; Counterfeit Drugs
• Other Study ID Numbers: STUDY00011759; 1R01HD103642-01 (U.S. NIH Grant/Contract)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: The de-identified analytic dataset and analytic R code will be made publicly available.
• IPD Sharing Time Frame: After publication.
• IPD Sharing Access Criteria: Publicly available
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ICF; ANALYTIC_CODE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: Yes