Effects of Probiotic in Treatment of Persistent Diarrhea in Children

Research of Change of Microbial System Intestines and Efficacy for Probiotic Treatment of Persistent Diarrhea in Children

Persistent diarrhea is a common health problem worldwide, particularly in low-income countries. Approximately 3% to 20% of acute diarrhea episodes in children under 5 years of age become persistent diarrhea. Persistent diarrhea causes malnutrition, weight loss, and dehydration, as well as increasing treatment costs and the risk of mortality. One of the main causes of persistent diarrhea is the overgrowth and spread of bacteria, as well as viral infections that can disrupt the balance of microorganisms in the gut. Antibiotics are effective in treating bacterial infections that cause persistent diarrhea in children, but not against viral or parasitic infections. Overuse of antibiotics can lead to the growth of antibiotic-resistant bacteria, which poses a significant public health concern. Probiotics play a vital role in maintaining a healthy balance. Bacillus probiotic strains have an advantage over Lactobacillus probiotics as they can form spores that resist environmental stressors like heat, acid, and bile. This makes them more likely to survive the harsh conditions of the digestive tract and provide health benefits by reaching the intestines intact. Here, the investigators propose high-dose Bacillus spore probiotic supplementation as a potential solution for treating patients with persistent diarrhea.

The aim of the study is to assess the efficacy of two types of Bacillus probiotics which conclude LiveSpo CLAUSY (2 billion B. clausii) and LiveSpo DIA 30 (5 billion B. subtilis, B. clausii and B. coagulans) in supporting the treatment of children with persistent diarrhea.

Study Population: sample size is 150 patients and 30 healthy children. The study is carried out at Vietnam National Children's Hospital.

Description of Study Intervention: Totally 150 eligible patients are divided randomly into 3 groups (n = 50/group each): Patients in the Control group received the routine treatment and 2-3 times/day RO water while the patients in the probiotics group received 2-3 times/day LiveSpo DIA 30 or LiveSpo CLAUSY in addition to the same standard of care treatment. The standard treatment regimen is 5-9 days but can be extended further depending on the severity of the patient. Healthy children are grouped into the "Healthy" group solely for the purpose of comparing the microbiota between healthy children with those patients before and after treatment. Therefore, the Healthy group does not receive any intervention.

Study duration: 18 months

Study Overview

• Status: Completed
• Conditions: Diarrhea
• Intervention / Treatment: Other: RO; Combination product: LiveSpo DIA30; Combination product: LiveSpo CLAUSY

Detailed Description

Persistent diarrhea, which is defined as diarrhea that lasts for more than two weeks but less than four weeks, is a widespread health concern globally, particularly in low-income countries. It is a significant public health issue for children, especially in regions such as Asia, South America, and Africa. According to the World Health Organization (WHO), diarrhea is the second leading cause of morbidity and mortality in children after acute respiratory infections. Persistent diarrhea is a significant problem that defeats the nutritional status of children and increases treatment costs. The common pathogens associated with persistent diarrhea include:

Bacteria: Escherichia coli (E. coli), Campylobacter jejuni, Yersinia, Salmonella Enteritidis, Shigella spp., Clostridium difficile, Aeromonas.

Parasite: Giardia lamblia, Entamoeba histolytica, Cryptosporidium, Cyclospora, Microsporidia.

Viruses: norovirus, rotavirus, adenovirus. The intestinal microbiota serves as a protective barrier against pathogenic microorganisms and their toxins, playing a crucial role in the development and regulation of the immune system within the gut. Alterations in the gut microbiota have been observed in children with persistent diarrhea, highlighting its importance in maintaining gut health.

In terms of persistent diarrhea treatment, antibiotics are effective in treating bacterial infections that cause persistent diarrhea in children, but not against viral or parasitic infections. Overuse of antibiotics can lead to the growth of antibiotic-resistant bacteria, which poses a significant public health concern. Probiotics are live microorganisms that can provide health benefits to the host when consumed in sufficient amounts, playing a vital role in maintaining a healthy balance, which typically range from 1 to 10 billion per day, depending on the intended purpose of either prevention or supportive treatment. High-dose Lactobacillus rhamnosus GG probiotics, up to 40 billion per day, have been shown to be safe and effective in the treatment of antibiotic-associated diarrhea However, the quality of evidence is moderate to low. In comparison to Lactobacillus species, Bacillus species, such as B. subtilis, B. clausii, and B. coagulans, have the ability to form spores, which are resistant to environmental stressors such as heat, acid, and bile. This means that Bacillus probiotics are more likely to survive the harsh conditions of the digestive tract and reach the intestines intact, where they can provide health benefits. Bacillus probiotics have been found to be naturally resistant to certain antibiotics, which may make them a more effective option for individuals who are taking antibiotics or who have a history of antibiotic use. Thus, the investigators propose high-dose Bacillus spore probiotic supplementation as a potential solution for treating patients suffering from persistent diarrhea.

The overall aim of this study was to to assess the efficacy of two types of probiotics, the first one containing 2 billion B. clausii spores (LiveSpo® CLAUSY) per ampoule, and the second one containing 5 billion spores of three bacterial species per ampoule (LiveSpo® DIA 30), including Bacillus subtilis, B. clausii, and B. coagulans, in supporting the treatment of children with persistent diarrhea.

The first objective is evaluating the effectiveness of these products in alleviating typical symptoms and reducing the duration of treatment. The secondary objective is measuring changes in stool properties, major cytokine and IgA indices in stool and blood samples, and microbiota composition before and after treatment with LiveSpo® DIA 30 and LiveSpo® CLAUSY.

For this aim, the study is designed as a randomized, blind, and controlled clinical trial with 150 participants diagnosed with persistent diarrhea. The participants are randomized into 3 groups using permuted block randomization: a control group using RO water, experimental group 1 using LiveSpo® DIA 30 probiotics, and experimental group 2 using LiveSpo® CLAUSY probiotics. Each patient received 4-6 ampoules of the assigned probiotic product daily, divided into 2-3 doses after meals. The study included daily clinical assessment of indicators such as the number of types of stool, presence of mucus in stool, and frequency of daily bowel movements.

Contents and Methods for sub-clinical detection. The study participants were children aged 2 to 24 months who exhibited signs of loose stools or abnormal water on more than 3 occasions per day for a duration of between 14 and 30 days, diagnosed with persistent diarrhea, with no other systemic illnesses except diarrhea upon admission to the hospital and during treatment.

• Hematology and biochemical tests are conducted on day 0 only as part of routine procedures at the Hematology and Biochemistry Department of the National Children's Hospital.
• A multiplex Real-time RT PCR assay is conducted on day 0 only at the Department of Molecular Biology for Infectious Diseases of the National Children's Hospital to detect 24 intestinal pathogens in stool samples for the purpose of consulting on appropriate treatment therapy.
• A real-time PCR assay is conducted on stool samples on day 0 and 5 discharge day at the Spobiotic Research Center (proper noun) to detect probiotic spores, including B. subtilis, B. clausii, and B. coagulans, to cross-check the proper usage of probiotics or placebo in the experimental and control groups, respectively.
• ELISA tests are conducted on stool and blood samples on day 0 and 5 discharge day at the Department of Molecular Biology for Infectious Diseases of the Vietnam National Children's Hospital to determine proinflammatory cytokine levels and IgA and TNF-alpha levels, respectively, for evaluating changes in immune-related indicators during the treatment.
• The 16S V3-V4 metagenome sequencing analysis was carried out at Macrogen (Seoul, Korea) using next-generation sequencing (NGS) technology on the Illumina MiSeq platform (Illumina, San Diego, CA, USA), utilizing a 2 × 250 bp run configuration. DNA extracted from stool samples of about 5-10 representative patients from each group on day 0 and day 3 or/and day 7 was used for the analysis, with the goal of identifying changes in the microbiome.
• Data collection and statistical analysis involve the collection of individual medical records and the systematization of patient information into a dataset.

The investigators recommend that the CLAUSY and DIA30 groups continue using probiotics at the prevention dosage of 1-2 ampoules per day for an additional 28 days after discharge. Afterward, conduct a telephone interview with the parents of patients in all three groups (Control, CLAUSY, and DIA30) to inquire about any typical symptoms of diarrhea that may have recurred on Day 28 (optional).

The tabular analysis is performed on dichotomous variables using the χ2 test or Fisher's exact test when the expected value of any cell is below five. Continuous variables are compared using either the Wilcoxon test, t-test, or the Mann-Whitney test when data are not normally distributed. To determine if there was a statistically significant difference between three groups, an ANOVA test was performed. The correlations among the variables are assessed by Spearman's correlation analysis. Statistical and graphical analyses are performed on GraphPad Prism v8.4.3 software (GraphPad Software, CA, USA). The significance level of all analyzes is set at p < 0.05. P-values. The efficacy of LiveSpo® DIA 30 and LiveSpo® CLAUSY is evaluated and compared to the control based on the following clinical and sub-clinical criteria obtained from the Experiment and Control groups: Primary outcome: (i) Symptomatic relief duration diarrhea; Secondary outcomes: (ii) Regulate in the levels of cytokines such as IL-6, IL-8, IL-10, IL-17, IL-23 and TNF-alpha in blood samples. (iii) Decrease in the IgA level in blood and fecal samples. (iv) Improvement in the diversity and count of beneficial bacterial species compared to harmful bacteria species in the gut microbiota.

• Study Type: Interventional
• Enrollment (Actual): 165
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Vietnam
  • Hanoi, Vietnam, 100000
    • Department of Gastroenterology, Vietnam National Children's Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Patients who are between 2 months and 24 months old
• Have loose or unusual watery stools more than 3 times per day, lasting between 14 and 30 days
• Parents of the pediatric patient agree to participate in the study, explain and sign the research consent form

Exclusion Criteria:

• The patient had any systemic illness other than diarrhea on admission.
• Patient has any systemic complications during treatment.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Triple

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Placebo Comparator: Control; Control group receives the routine treatment and uses distilled water (RO) water Routine treatment at Department of Gastroenterology, Vietnam National Children's Hospital is as follows: Antibiotics: oral (e.g. Zithromax® or Ciprofloxacin®) or intervention (ceftriaxone®, Ciprofloxacin®, Metronidazole®, Vancomycin®) drugs.; Oral rehydration Solution: Oremute; Zinc gluconate	Other: RO; Aquafina's distilled water and reverse osmosis (RO) water, produce by PepsiCo, have both obtained ISO 9001:2015 and ISO 22000:2018 certifications, which are internationally recognized standards for quality management and food safety management systems, respectively. The RO water ampoules are produced using a similar process as the LIVESPO DIA30/CLAUSY but contain 5ml of high-quality distilled water from Aquafina in an opaque plastic bottle
Experimental: DIA30; DIA 30 group receives the routine treatment and uses distilled water plus B. subtilis, B. clausii and B. coagulans at 5 billion CFU/5 mL (LiveSpo® DIA 30) Routine treatment at Department of Gastroenterology, Vietnam National Children's Hospital is as follows: Antibiotics: oral (e.g. Zithromax® or Ciprofloxacin®) or intervention (ceftriaxone®, Ciprofloxacin®, Metronidazole®, Vancomycin®) drugs.; Oral rehydration Solution: Oremute; Zinc gluconate	Combination product: LiveSpo DIA30; LiveSpo® DIA 30 has a registration number: 6547/2019/ĐKSP issued by the Food Safety Department of the Ministry of Health in Vietnam
Experimental: CLAUSY; CLAUSY group receives the routine treatment and uses distilled water plus B. clausii at 2 billion CFU/5 mL (LiveSpo® CLAUSY) Routine treatment at Department of Gastroenterology, Vietnam National Children's Hospital is as follows: Antibiotics: oral (e.g. Zithromax® or Ciprofloxacin®) or intervention (ceftriaxone®, Ciprofloxacin®, Metronidazole®, Vancomycin®) drugs.; Oral rehydration Solution: Oremute; Zinc gluconate	Combination product: LiveSpo CLAUSY; LiveSpo® CLAUSY has a registration number: 4071/2021/ĐKSP issued by the Food Safety Department of the Ministry of Health in Vietnam

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in days of treatment for typical symptoms of persistent diarrhea	Changes in days of treatment for typical symptoms of persistent diarrhea, including: the frequency of bowel movements per day (times/day); the presence of mucus in stool (2 - lots of mucus, 1 - less mucus, 0 - no mucus); the Bristol Stool Score ( 1 - Type 1, 2 - Type 2, 3 - Type 3, 4 - Type 4, 5 - Type 5A, 6 - Type 5B)	Day 0 to Day 12

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in presence of white blood cells and red blood cells by stool microscopy	Changes in presence of white blood cells (yes/no) and red blood cells (yes/no).	Day 3 and Day 5 compared to Day 0
Changes in Intestinal microbiota	Changes in intestinal microbiota (bacterial species composition) in the stool samples of patients with persistent diarrhea before (Day 0) and after treatment (Day 3 or/and 7), as well as with that of healthy children (Day 0, no intervention)	Day 3 or/and Day 7 compared to Day 0
Changes in cytokines levels of blood samples	Changes in levels (pg/mL) in several cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-17 (IL-17), interleukin-23 (IL-23)	Day 5 compared to Day 0
Changes in IgA levels in both stool and blood samples	Changes in levels (µg/mL) of pro-inflammatory IgA in both stool and blood samples	Day 5 compared to Day 0
Change the stool pH values	Change the pH values of stool samples at day 3-5 (after treatment) compared with day 0 (before treatment)	Day 3 and Day 5 compared to Day 0

Collaborators and Investigators

• Sponsor: National Children's Hospital, Vietnam
• Investigators: Principal Investigator: Dang T Ha, MD, Department of Gastroenterology, Vietnam National Children's Hospital

Publications and helpful links

General Publications

• Szajewska H, Kolodziej M. Systematic review with meta-analysis: Lactobacillus rhamnosus GG in the prevention of antibiotic-associated diarrhoea in children and adults. Aliment Pharmacol Ther. 2015 Nov;42(10):1149-57. doi: 10.1111/apt.13404. Epub 2015 Sep 13.
• Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014 Aug;11(8):506-14. doi: 10.1038/nrgastro.2014.66. Epub 2014 Jun 10.
• Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014 Mar 27;157(1):121-41. doi: 10.1016/j.cell.2014.03.011.
• Sarker SA, Ahmed T, Brussow H. Persistent diarrhea: a persistent infection with enteropathogens or a gut commensal dysbiosis? Environ Microbiol. 2017 Oct;19(10):3789-3801. doi: 10.1111/1462-2920.13873. Epub 2017 Sep 14.
• Fan Q, Yi M, Liu H, Wang Y, Li X, Yuan J, Wang L, Hou B, Li M. The Impact of Age and Pathogens Type on the Gut Microbiota in Infants with Diarrhea in Dalian, China. Can J Infect Dis Med Microbiol. 2020 Nov 30;2020:8837156. doi: 10.1155/2020/8837156. eCollection 2020.
• Bandsma RHJ, Sadiq K, Bhutta ZA. Persistent diarrhoea: current knowledge and novel concepts. Paediatr Int Child Health. 2019 Feb;39(1):41-47. doi: 10.1080/20469047.2018.1504412. Epub 2018 Aug 6.
• Ugboko HU, Nwinyi OC, Oranusi SU, Oyewale JO. Childhood diarrhoeal diseases in developing countries. Heliyon. 2020 Apr 13;6(4):e03690. doi: 10.1016/j.heliyon.2020.e03690. eCollection 2020 Apr. Erratum In: Heliyon. 2020 Jun 10;6(6):e04040. doi: 10.1016/j.heliyon.2020.e04040.
• Abba K, Sinfield R, Hart CA, Garner P. Pathogens associated with persistent diarrhoea in children in low and middle income countries: systematic review. BMC Infect Dis. 2009 Jun 10;9:88. doi: 10.1186/1471-2334-9-88.
• Olvera-Rosales LB, Cruz-Guerrero AE, Ramirez-Moreno E, Quintero-Lira A, Contreras-Lopez E, Jaimez-Ordaz J, Castaneda-Ovando A, Anorve-Morga J, Calderon-Ramos ZG, Arias-Rico J, Gonzalez-Olivares LG. Impact of the Gut Microbiota Balance on the Health-Disease Relationship: The Importance of Consuming Probiotics and Prebiotics. Foods. 2021 Jun 2;10(6):1261. doi: 10.3390/foods10061261.
• Guo Q, Goldenberg JZ, Humphrey C, El Dib R, Johnston BC. Probiotics for the prevention of pediatric antibiotic-associated diarrhea. Cochrane Database Syst Rev. 2019 Apr 30;4(4):CD004827. doi: 10.1002/14651858.CD004827.pub5.
• Tridip K. Das, Shrabani Pradhan, Sudipta Chakrabarti, Keshab Chandra Mondal, Kuntal Ghosh, Current status of probiotic and related health benefits, Applied Food Research, Volume 2, Issue 2, 2022, 100185, ISSN 2772-5022

Helpful Links

• Diarrheal disease

Study record dates

Study Major Dates

• Study Start (Actual): July 1, 2022
• Primary Completion (Actual): December 25, 2023
• Study Completion (Actual): November 25, 2024

Study Registration Dates

• First Submitted: March 25, 2023
• First Submitted That Met QC Criteria: April 12, 2023
• First Posted (Actual): April 14, 2023

Study Record Updates

• Last Update Posted (Estimated): December 10, 2024
• Last Update Submitted That Met QC Criteria: December 5, 2024
• Last Verified: July 1, 2024

More Information

Terms related to this study

• Keywords: Children; Gut microbiota; Viruses; Bacterial; Persistent diarrhea; Bacillus spore
• Additional Relevant MeSH Terms: Signs and Symptoms, Digestive; Diarrhea
• Other Study ID Numbers: 9720109

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: Data or samples share that will be coded, with no PHI include. Approval of the request and execution of all applicable agreements (i.e. a material transfer agreement) are prerequisites to the sharing of data with the requesting party.
• IPD Sharing Time Frame: Data requests can be submitted starting 9 months after article publication and the data will be made accessible for up to 24 months. Extensions will be considered on a case-by-case basis
• IPD Sharing Access Criteria: Access to trial IPD can be requested by qualified researchers engaging in independent scientific research and will be provided following review and approval of a study protocol, informed consent form (ICF), clinical study report (CSR). For more information or to submit a request, please contact clinicaltrial.probiotics@gmail.com
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ICF; CSR

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.: No