Received: 6 November 2020 Revised: 15 March 2021 Accepted: 14 June 2021

DOI: 10.1111/pedi.13244

ORIGINAL ARTICLE

A high potency multi-strain probiotic improves glycemic

control in children with new-onset type 1 diabetes mellitus: A
randomized, double-blind, and placebo-controlled pilot study

Sanjeev Kumar1 | Rakesh Kumar1 | Latika Rohilla1 | Neenu Jacob1 |

1 2
Jaivinder Yadav | Naresh Sachdeva

1
Department of Pediatrics, Post-Graduate
Institute of Medical Education and Research
(PGIMER), Chandigarh, India
2
Department of Endocrinology, Post-Graduate
Institute of Medical Education and Research
(PGIMER), Chandigarh, India
Correspondence
Dr. Rakesh Kumar, Professor, Endocrinology
and Diabetes Unit, Department of Pediatrics,
Room No 3117, Level 3, A Block, Advanced
Pediatrics Centre,Post Graduate Institute of
Medical Education and Research (PGIMER),
Sector 12, Chandigarh 160012, India.
Email: drrakesh.pgi@gmail.com
Abstract
Background: Studies in animal models and humans with type 1 diabetes mellitus
(T1DM) have shown that probiotic supplementation leads to decreased pro-
inflammatory cytokines (responsible for damaging β-cells of the pancreas), improved
gut barrier function, and induction of immune tolerance.
Objective: To study the effect of supplementation of probiotics in children with
T1DM on glycemic control, insulin dose, and plasma C-peptide levels.
Methods: A single-centered, double-blinded, and randomized placebo-controlled pilot
trial was conducted in children (2–12 years) with new-onset T1DM. Ninety-six children
were randomized and allocated to Placebo or Intervention groups. The intervention
included high dose (112.5 billion viable lyophilized bacteria per capsule) multi-strain probi-
otic De Simone formulation (manufactured by Danisco-Dupont) sold as Visbiome® in
India. The probiotic was supplemented for 3 months and HbA1c, fasting C-peptide, blood
sugar records, and insulin dose was recorded at baseline and 3 months.
Results: A total of 90 patients (45 in each group) were analyzed for outcome parame-
ters. We found a significant decrease in HbA1c (5.1 vs. 3.8; p = 0.021) and a signifi-
cant decline in total and bolus insulin dose (U/kg/day; p = 0.037 and 0.018,
respectively) in the intervention group when compared with the placebo group. A sig-
nificantly higher (p = 0.023) number of children achieved remission in the treatment
group. We did not notice adverse effects in either of the study groups.
Conclusion: Children with newly diagnosed T1DM managed with standard treatment
along with probiotics showed better glycemic control and a decrease in insulin
requirements; however, more extensive studies are further warranted.

KEYWORDS
blood glucose, type 1 diabetes mellitus, double-blinding, insulin, probiotics

1 | I N T RO DU CT I O N until approximately 80% has been destroyed, following which hyper-

glycemia initiates. Complete loss of β-cells gradually leads to signifi-
Type 1 diabetes mellitus (T1DM) is a debilitating autoimmune disorder cant insulin deficiency, worsening hyperglycemia symptoms, and the
resulting in cytotoxic T-cell mediated destruction of the pancreatic absolute necessity of exogenous insulin administration. The preva-
insulin-producing β-cells.1 The β-cells destruction remains subclinical lence of T1DM varies geographically,2 and it is rising in some

Pediatr Diabetes. 2021;1–9. wileyonlinelibrary.com/journal/pedi © 2021 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd 1
2 KUMAR ET AL.
regions.3T1DM is one of the most common chronic metabolic dis-
eases of childhood. The global incidence is increasing by 3%–5%
4
every year, with India accounting for most cases in South Asia.
The pathophysiology of T1DM is multifactorial; both genetic pre-
disposition and nongenetic factors have been implicated in its patho-
genesis. Although genetically predisposed individuals possess a higher
2
risk for the disorder, less than 10% develop the disease. While
genetic factors like polymorphisms in specific alleles (HLA DR4-DQ8
and DR3-DQ2) could make a child susceptible to develop T1DM, the
focus has shifted to nongenetic factors like environmental influences,
which may also be responsible for the rising incidence of this disor-
der.5 In addition to the genetic and environmental factors, a linkage
between disarrayed intestinal flora, a faulty intestinal mucosal barrier,
and altered mucosal immunity has been shown to contribute to the
pathogenesis of the disease.6 Many toll-like receptors (TLRs), includ-
ing macrophages, dendritic cells (DCs), specific T-cells, and natural
killer cells produced by immune cells, which along with cytokines, such
as IL-1β, IL-12, IFN-γ, and TNF-α have been reported to cause direct
damage to pancreatic β-cells. 7,8
Probiotics are defined as live microorganisms, which when
ingested in adequate amounts, confer a health benefit on the host.9
The gut microbiome has been reported to be an essential environmen-
tal factor in the development of T1DM. The gut microflora of healthy
individuals differs considerably from those of prediabetics or in
patients with established diabetes. Although the exact mechanism of
altered gut microbiota modulating the development of T1DM is not
fully understood, susceptibility to T1DM has been linked to the
changes in the composition of gut microbiota, especially with an
increased number of bacteria belonging to the Bacteroidetes phylum
and a corresponding decrease in the number of Lactobacillus,
Bifidobacterium and Clostridium strains.10 It is hypothesized that alter-
ations in gut microbiota in the early years of life could induce
immune-intolerance leading to antipancreatic antibody development
in the first few years of life auto-immunity progresses to T1DM due
to reduced microbial diversity and a pro-inflammatory intestinal
dysbiosis.11 The results from a recent TEDDY study, analyzing longitu-
dinal stool samples of 903 (genetically at-risk of T1DM) infants, have
shown that the gut microbiome evolves till 3 years of life when it is
relatively stable both in pancreatic antibody positive/T1DM and
healthy control toddlers with comparable alpha diversity. However,
toddlers developing T1DM had higher levels of Streptococcus sp. and
Lactococcus sp., and lower levels of Akkermansia.12 Another TEDDY
study cohort of 783 children (case–control) assessed various aspects
of gut microbiota longitudinally until the endpoint of development of
pancreatic antibodies or T1DM reported that short-chain fatty acids
producing gut bacteria were protective against T1DM and were seen
13
to be more abundant among healthy controls.
A good association has been reported between probiotic con-
sumption and metabolic profile in diabetic individuals.14 The concept
of manipulating the gut microbiota has gained considerable impor-
tance in recent years. Several studies on probiotics propose different
mechanisms to prevent or delay the onset of T1DM, such as increased
glucagon-like peptide-1 (GLP-1) secretion to improve carbohydrate
metabolism, decreased glucotoxicity,15 improved integrity of intestinal
epithelium, and suppressed TLR pathway. Moreover, few studies have
shown the effect of probiotics in reducing pro-inflammatory signals
and enhancing insulin sensitivity, altering several genes' expression on
preventions or delaying the onset of T1DM.14,16–18 Though there has
been extensive research in animal studies, the studies assessing the
effects of probiotics in T1D children are minimal. Therefore, this pilot
study was conducted to determine the impact of high potency, multi-
strain probiotics on glycemic control in children with new-
onset T1DM.
2 | METHODS
2.1 | Study design
This investigator-initiated study was conducted as a randomized,
double-blind placebo-controlled, pilot clinical trial at a tertiary care
hospital in Northern India, Post-Graduate Institute of Medical Educa-
tion and Research (PGIMER), Chandigarh from October 2017 to
November 2018. The Institute Ethics Committee approved the study
(Letter No. INT/IEC/2017/390 dated April 08, 2017) and was con-
ducted in conformance to the 1975 Declaration of Helsinki's ethical
guidelines. The trial protocol was registered prospectively with Clinical
Trial Registry India (CTRI); No. CTRI/2017/06/008725 (Supplemen-
tary File 1). Informed consent was obtained from all study partici-
pants. The investigational drugs were supplied by NextGen Pharma
India Pvt. Ltd., New Delhi, but the company did not participate in any
study-related activities or decisions.
2.2 | Participants
Children between 2 and 12 years of age with new-onset T1DM
(within 6 months of enrollment) and registered in 'Pediatric Diabetes
Clinic' were screened for enrollment after obtaining informed consent
from parents or legal guardians. Children with diarrhea, chronic renal/
liver diseases, acute infective or systemic inflammatory illnesses, and
chronic medical conditions known to influence gut microbiota (ulcera-
tive colitis, irritable bowel syndrome, and Crohn's disease, etc.) were
excluded. Children with a positive screening test for celiac disease or
children on antibiotics, prebiotics, probiotics, symbiotics, or laxatives
in the past 15 days were also excluded.
2.3 | Clinical and laboratory assessments
Demographic characteristics, medical/surgical history, and current
medications were recorded at baseline. Clinical evaluation included a
thorough general physical examination. Laboratory investigations
included a complete hemogram, serum biochemistry (including a lipid
profile, thyroid profile, renal and liver function tests), random, glycated
hemoglobin (HbA1c), and determination C-peptide levels. Each
KUMAR ET AL.
participant was provided with the choice for insulin injection, with a
preference for the anterior abdominal wall for bolus and thigh for
basal insulin. Insulin type, total dose, basal dose, and unmodified insu-
lin dose at baseline were recorded. Participants were instructed to
monitor and record blood glucose levels at least four times a day
(fasting, prelunch, predinner, and bedtime). The participants were
advised to record any diabetes-related or gastrointestinal adverse
reactions, such as frequency of hypoglycemic episodes (asymptomatic,
symptomatic, severe symptomatic or nocturnal), episodes of diabetic
ketoacidosis (DKA), or flatulence. The recruited participants were pro-
vided with the investigational drugs for 3 months and were instructed
to visit the clinic at the end of 3 months. Compliance was ensured by
a biweekly telephone call to the caregivers and collecting and cou-
nting the study drug's empty packs. Telephonic follow-up was also
used to document any problems related to the prescribed medication
if any.
At the concluding visit, clinical examination and laboratory
investigations were re-performed. Anthropometric measurements
and assessment of insulin regimen, frequency of DKA, and hypo-
glycemic episodes were assessed. Self-monitored blood glucose
records were collected. Data from seven consecutive days before
the study visit were averaged to calculate mean fasting blood glu-
cose and mean blood glucose levels. Glucose variability (GV) (SD/
mean glucose  100 percentage) was calculated on glucose values
over seven consecutive days before each assessment. The plasma
C-peptide levels were determined by electro-chemiluminescence
immuno-assay (ELECSYS-2010, Roche Diagnostics) as per the
manufacturer's protocol using the kits, controls and calibrators
supplied by the manufacturer. HbA1c was determined as per the
manufacturer's protocol using an automated HPLC based system
with ion exchange resin, control and calibrators provided by the
manufacturer (Variant II Turbo, BioRad, USA).
2.4 | Randomization and intervention
2.4.1 | Randomization, blinding, and allocation
concealment
Computer-generated randomization using permuted blocks of
10 was prepared by an independent person, not involved in the
study. The master randomization list was blinded till the end of the
study. According to the pharmaceutical company's randomization
schedule, the investigational drugs (active and placebo) as identical
blisters containing same-colored capsules were prepacked in enve-
lopes and consecutively numbered for each participant. Each par-
ticipant was assigned the same number corresponding to the
number marked on the prepacked envelope. The allocation
sequence was concealed from the investigators, participants, and
outcome assessors in sequentially numbered, opaque, sealed, and
stapled envelopes. The investigators and participants were
instructed to store the investigational drugs at 2–8 C till
consumed.
3
2.4.2 | Intervention
Patients were randomized in a double-blind fashion to receive either a
high-potency, multi-strain probiotic preparation or a placebo for
3 months. All participants were instructed to ingest one capsule
(or contents of the capsule for younger children) with cold water
before their evening meal.
Probiotic group
One capsule orally daily (Each capsule was containing 112.5 billion
live, lyophilized, lactic acid bacteria and bifidobacteria, namely L para-
casei DSM 24733, L plantarum DSM 24730, L acidophilus DSM 24735,
and L delbrueckii subsp. bulgaricus DSM 24734, B longum DSM 24736,
B infantis DSM 24737, B breve DSM 24732, and Streptococcus
thermophilus DSM 24731 sold in Europe as VIVOMIXX® and
VISBIOME® in India (De Simone formulation).
Placebo group
One capsule orally daily (Capsule contained microcrystalline cellulose).
2.5 | Outcome measures
The study's primary objective was to assess the changes in HbA1c
levels. The secondary objectives were to determine changes in C-
peptide levels, insulin dose, and blood GV in children with new-onset
T1DM after 3 months of probiotic supplementation. The HbA1c was
taken as a primary outcome measure (rather than C-peptide) as we
could not fix the sampling timing for C-peptide (in relation to meals).
The patients had to come from far off places to our out-patient
department with variable time since the last meal, which could have
altered the serum C-peptide values.
2.6 | Concomitant diet and medications
All participants were prescribed a meal plan based on local/family
preferences, consisting of three meals and either two or three snacks
as per the formal instructions from a dietitian about the principles and
application of the diet for children with T1DM.
The following medications/interventions were permitted during
the study: Insulin dose was titrated as per need in each group. Other
permitted medications included levothyroxine and vitamin D replace-
ment if warranted. Any patient requiring the use of antibiotics more
than 5 days (for any infection) during the treatment period was
dropped out of the study.
2.7 | Sample size calculation and statistical
methods
The study was planned based on an assumption of a typical SD of
HbA1c as 1.5% with a 1:1 randomization ratio. Sample size
4 KUMAR ET AL.
calculations by considering an absolute difference (δ) of 1% in HbA1c
between study groups, with an alpha error of 0.05 (two-sided) and a
beta error of 0.20, using the formula (n = 2 * K *SD2/δ2; where K is
constant and δ is the magnitude of difference to be detected), gave
the results as 45 participants per arm. Considering an expected attri-
tion rate of 10%, five extra participants were planned for recruitment
in each group.
Continuous data were expressed as mean ± SD/median and inter-
quartile range (IQR), and categorical variables were expressed as per-
centages. The normality of the distribution was assessed using the
Kolmogorov–Smirnov test. Variables with a normal distribution were
presented as mean ± SD, and variables with a skewed distribution
were presented as the median (IQR). Baseline descriptive data
between the control and intervention groups were compared using
chi-square for categorical variables and t-tests for continuous vari-
ables. The primary outcome variable HbA1c was expressed as both
mean ± SD and Median (IQR) as its data was slightly skewed in distri-
bution. Both two-sided t-test and Wilcoxon signed-rank test were
used separately to compare HbA1c between the two groups. Differ-
ences between the treatment groups for C-peptide levels, total
insulin, basal insulin, and bolus insulin were compared using a two-
sided t-test and Wilcoxon signed-rank test as applicable. Between and
within-group comparisons at baseline and follow-up visits were con-
ducted using the Wilcoxon signed-rank test and Mann–Whitney test.
Both intention-to-treat (ITT) and per-protocol (PP) analysis were per-
formed for main outcome variables. The PP analysis was presented
for the sake of convenience, as there was no difference in both ana-
lyses. p values of less than 0.05 was considered statistically significant.
The analysis was conducted using SPSS for Windows (version20.0;
SPSS Inc., Chicago, IL, USA).
3 | RESULTS
3.1 | Participant flow
Of 117 patients screened for inclusion in the study, we could not
include 21 (seven did not meet inclusion criteria, five were excluded
as per exclusion criteria, and nine did not consent to participate in the
study). A total of 96 patients were randomized to receive either probi-
otic (n = 47, 49%) or placebo (n = 49, 51%) (CONSORT diagram as
Figure 1). Forty-five patients each in the probiotic group and the pla-
cebo group completed the study. Six patients were excluded from the
study analysis (PP) as they failed to undergo investigations at the
follow-up. The CONSORT checklist is provided as Supplementary
File 2.
3.2 | Baseline characteristics
The baseline characteristics of all enrolled patients are shown in
Table 1. All baseline characteristics, except total T3 and T4 and GV,
were comparable between the two groups. Serum total T3 and T4
were significantly higher (p value = 0.010 and 0.017 respectively) in
the placebo group. The GV in fasting blood glucose and mean glucose
levels were significantly higher in the probiotic group at the baseline.
3.3 | Primary and secondary outcomes
The delta change in the primary and secondary outcome measures at
3 months is shown in Table 2. A significantly higher decline in HbA1c
levels was observed in the probiotic group than the placebo group
(p = 0.021, PP; p = 0.012, ITT) at the end of 3 months. The total and
bolus insulin dose (U/kg/day) showed a significantly higher reduction
in the probiotic group than the placebo group (p = 0.0037 and
p = 0.018 respectively, PP; p = 0.02 and p = 0.017 respectively, ITT)
at the end of 3 months. There was no significant difference in the C-
peptide levels (p = 0.971) and basal insulin requirement (p = 0.842)
between the two groups. The fall in GV between the two groups was
also significant after 3 months of treatment. The fall in GV of all blood
sugars recorded over the last week before the follow-up was signifi-
cantly more (p < 0.001) in the probiotic group. However, the fall in GV
of fasting blood sugars favored the placebo group at the end of
3 months.
Comparison of the outcome parameters at baseline and 3 months
is shown in Table 3. Improvements in levels of HbA1c and insulin
requirements were seen in both groups. One participant in both the
probiotic and placebo group had become off-insulin after 3 months
due to remission. The number of children achieving remission over
the study period (defined as requirement of insulin <0.5 U/Kg/day
and HbA1c <7%) was significantly higher (p = 0.023) in the probiotic
group (n = 12, 26.6%) as compared to the children in the placebo
group (n = 4, 8.8%).
More participants in the probiotic group showed a reduction in
HbA1c by more than 50% than the baseline at 3 months from baseline
compared to the placebo participants (14 vs. 8, p = n.s.). Fourteen (31.1%)
and 17 (39.5%) participants in the probiotic group showed a significant
change of 50% or more in total insulin and bolus insulin requirement as
compared to 5 (11.4%) in the placebo group (p = 0.001 and p < 0.001
respectively) (Supplementary File 3; Table S1).
Four (8.9%) patients in the placebo group showed an 'increase' in
HbA1c as compared to 1 (2.2%) in the probiotic group after 3 months
of supplementation (p = 0.018). Similarly, 14 (31.8%), 18 (40.9%) and
13 (30.2%) in the placebo group showed an 'increase' in requirements
of total insulin, basal insulin and bolus insulin as compared to 4 (9.3%),
10 (23.3%), and 5 (11.9%) in the probiotic group after 3 months of
supplementation (p < 0.001, p = 0.026, and p = 0.003), respectively.
There were no significant changes in C-peptide levels at 3 months
(Supplementary File 3; Table S2).
3.4 | Adverse events
The investigational drugs were well tolerated. Mild adverse events like
bloating and flatulence were reported in 2 (4.4%) participants in the
KUMAR ET AL. 5

FIGURE 1 Flow of patients

T A B L E 1 Participants baseline
Baseline parametersa Probiotic (n = 47) Placebo (n = 49) p value
characteristics
Age in years 7.92 9.10 0.102d
Median (IQR) (3.92)b (4.95)b
Male, n (%) 28 (59.6) 28 (57.1) 0.743c
Duration of Diabetes (days) 46 56 0.414c
Median (IQR) (38)b (132)b
DKA at Dx, n (%) 9 (19.1) 11 (22.4) 0.691c
BMI in kg/m2 15.2 ± 1.8 15.7 ± 2.3 0.225c
BMI Z-score 0.6 ± 1.1 0.5 ± 1.3 0.879c
TSH (μIU/ml) 2.6 ± 1.4 2.7 ± 1.4 0.685c
T4 (μg/dl) 6.3 ± 1.7 7.2 ± 1.7 0.017c
T3 (ng/dl) 1.0 ± 0.3 1.3 ± 0.5 0.010c
Mean FBG over last 7 days (mg/dl) 133.8 ± 57.5 124.3 ± 39.4 0.363c
Mean blood glucose over last 7 days (mg/dl) 161.6 ± 59.8 147.3 ± 36.0 0.172c
b b
Baseline HbA1c (g%) 11.7 (2.8) 11.5 (3.9) 0.130d
C-peptide (ng/ml)) 0.3 (0.5)b 0.3 (0.7)b 0.971d
b b
Total insulin dose (U/Kg/Day) 1.0 (0.7) 0.9 (0.6) 0.376d
Basal insulin dose (U/Kg/Day) 0.3 (0.1)b 0.3 (0.2)b 0.842d
b b
Bolus insulin dose (U/Kg/Day) 0.7 (0.5) 0.6 (0.4) 0.141d
GV in FBG over past 7 days (%) 37.0 31.6b 0.001c
GV in all blood glucose over past 7 days (%) 59.8 24.4 <0.001c

Abbreviations: BMI, body mass index; DKA, diabetic ketoacidosis; FBG, fasting blood glucose; GV,
glucose variability; HbA1c, glycated hemoglobin; T3, triiodothyronine; T4, thyroxine; TSH, thyroid-
stimulating hormones.
a
except where indicated data are presented as mean ± SD.
b
data presented as Median (IQR).
c
student t test.
d
Wilcoxon-signed rank test.
6 KUMAR ET AL.

TABLE 2 Outcome measures at 3 months in two groups

Outcome measures/arms Probioti (n = 45) Placebo (n = 45) p value (PP)

a a
Change in HbA1c (g%) 5.1 (3.9) 3.8 (5.3) 0.021f
Change in C-peptide (ng/ml) 0.2 (0.5)a 0.2 (0.5)a 0.901f
b a a
Decline in total insulin requirement (U/Kg/day) 0.3 (0.6) 0.1 (0.4) 0.037f
Decline in basal insulin requirement (U/Kg/day)c 0.1 (0.2)a 0.0 (0.1)a 0.086f
d a a
Decline in bolus insulin requirement (U/Kg/day) 0.2 (0.6) 0.1 (0.3) 0.018f
Fall in GV in FBG over past 7 days (%) 6.5 8.2 <0.001e
Fall in GV in all blood glucose over past 7 days (%) 18.7 2.9 <0.001e

Abbreviations: FBG, fasting blood glucose; HbA1c, glycated hemoglobin; PP, per protocol.
a
data presented as Median (IQR).
b
one participant in each group was off insulin.
c
one additional participant was off basal insulin requirement on probiotic group.
d
one additional participant was off bolus insulin requirement on the probiotic group.
e
student t test.
f
Wilcoxon signed-rank test.

TABLE 3 Comparison of parameters at baseline and 3 months in two groups

Probiotic Placebo

Baseline 3 mon Baseline 3 mon

Parametersh /Arms (n = 47) (n = 45) p valueb (n = 49) (n = 45) p valuec p valuea
HbA1c (g%) .7 (2.8)d 6.8 (2.5)d <0.001e 11.5 (3.9) 7.4 (2.5) <0.001e 0.512f
C-peptide (ng/ml) 0.3 (0.5)d 0.5 (0.6)d 0.025e 0.3 (0.7) 0.5 (0.5) 0.038e 0.866f
d d e e
Total insulin dose (U/Kg/day) 1.0 (0.7) 0.6 (0.4) <0.001 0.9 (0.6) 0.7 (0.6) 0.001 0.183f
Basal insulin dose (U/Kg/day) 0.3 (0.1)d 0.2 (0.2)d 0.004e 0.3 (0.2) 0.3 (0.2) 0.181e 0.131f
d d e e
Bolus insulin dose (U/kg/day) 0.7 (0.5) 0.4 (0.3) <0.001 0.6 (0.4) 0.5 (0.4) 0.002 0.288f
Mean glucose variability in FBG over past 7 days 37.0 43.5 0.472g 31.6 23.4 0.370g <0.001g
g g
Mean glucose variability in mean blood 59.8 41.1 0.735 24.4 21.5 0.255 <0.001g
glucose over past 7 days
a
Between-group comparison at 3 mon (probiotic group vs. placebo group),
b
Within-group comparison (probiotic group; 0 vs 3 mon).
c
Within-group comparison (placebo group; 0 vs. 3 mon).
d
Data presented as Median (IQR).
e
Mann Whitney Test.
f
Wilcoxon signed-rank test.
g
Student t test.
h
Except where indicated data are presented as mean ± SD.

probiotic group. Participants on the placebo did not have any com-
plaints throughout the study.
4 | DISCUSSION
In this randomized, double-blind, placebo-controlled pilot study, we
explored the role of a high potency multi-strain probiotic preparation
primarily on glycemic control in children with new-onset T1DM. Par-
ticipants in the probiotic group demonstrated a significant fall in
HbA1c levels after 3months of supplementation. Though no changes
in C-peptide levels were observed, total and bolus insulin require-
ments were reduced significantly in the probiotic group compared to
the placebo group. The results of this preliminary study suggest a
potential benefit of probiotics on glycemic control, further strengthen-
ing the hypothesis that gut dysbiosis has a role to play in the patho-
genesis of insulin deficiency and T1DM. Our study was not powered
to look for differences in C-peptide in the two groups and future stud-
ies with a larger sample size would be needed with to explore the
effect of probiotics on C-peptide levels.
A few recent studies have suggested that probiotics could be use-
ful in preventing the development of auto-immunity or T1DM in
(genetically) at-risk individuals.12,13 A large study by Uusitalo U et al.
exploring the Association of Early Exposure of Probiotics and Islet
Autoimmunity in the TEDDY Study concluded that early exposure to
probiotics (0–27 days after birth) could significantly reduce the risk of
developing islet autoimmunity in the future (hazard ratio of 0.66; 95%
CI 0.45–0.96).18
KUMAR ET AL.
The clinical trials assessing the effect of probiotics in individuals with
T1DM are sparse. We could identify very few trials evaluating the role of
pre or probiotics in glycemic control among individuals with T1DM. An
19
RCT conducted by Ho et al. in 43 newly diagnosed (within 3 months of
diagnosis) T1DM children and adolescents (8–17 years), with 38 children
completing the trial. Oligo fructose-enriched inulin (prebiotic with a dose
of 8 gm/day) or placebo was supplemented for 12 weeks study period.
There was no significant difference in mean HbA1c levels and other gly-
cemic control parameters between the two groups (although fall was
more in the prebiotic group). The authors also reported significant preser-
vation of C-peptide levels at the end of 3 months in the prebiotic group.
Our study provided probiotics rather than prebiotics and a high strength
over the same duration of 3 months, which could explain the more
remarkable Improvement in HbA1c. Another RCT performed by Javid
20
et al. enrolled 50 children and adolescents (4 to 18 years) with TIDM
(within 1 year of diagnosis), of which 44 children completed the study.
9
The synbiotic powder containing 10 CFU of Lactobacillus sporogenes
GBI-30 (probiotic), maltodextrin, and fructo-oligosaccharide (prebiotic) or
placebo was given for 8 weeks at 2 gm per day. There was a significant
reduction in mean fasting blood sugar and mean HbA1c in the interven-
tion group at the end of 8 weeks. A recent study by Adly et al. (presented
as an abstract in ISPAD 2020 meeting) also reported a significant glyce-
mic improvement in children with T1DM (for more than 1-year duration)
after a 6-month supplementation of probiotics (Lactobacillus acidophilus
La-14 [108CFU]), as compared to control group. This open-label trial
included 70 children, including controls, between 5–18 years. The study
also documented the immune-modulatory effects of probiotics on IL-21
and IL-22 and concluded that probiotics could be an adjuvant treatment
for children with T1DM.21 All the above studies have shown Improve-
ment or trend towards improved glycemic control in groups receiving
9
pre/probiotics even in a lower dose (10 CFU) or single strain. However,
in our study, we used high-strength multi-strain probiotics containing
1011CFU of bacteria.
We did not find any significant differences in blood C-peptide
levels among the two groups at the end of 3 months. However, C-
peptide levels improved in both groups (likely related to partial remis-
sion). Further, there was no improvement in fasting blood sugars and
any significant reduction in basal insulin dose, both of which usually
correlates with individuals' background insulin secretion or beta-cell
reserve. So, the exact mechanism by which probiotics improved the
glycemic profile is not precise. Ho et al.19 reported significant
Improvement in C-peptide after 3 months of prebiotic supplementa-
tion (but did not show substantial glycemic Improvement). Javid
et al.20 did not measure and report C-peptide levels in their study.
However, there are some other reported mechanisms for pre or pro-
biotics to improve the glycemic profile. It has been suggested that pre
22
and probiotics can improve serum lipid levels and insulin resistance.
Also, Synbiotics can increase GLP-1 and GLP-2 secretion, which in
turn can lead to loss of weight, lower blood glucose, and improved
HbA1c.23
The exact mechanisms whereby intestinal bacteria are altered
and how such alterations influence T1DM development are still
explored.24 Multiple mechanisms by which probiotics can confer
7
glycemic control have been proposed in various studies, namely,
Improvement in insulitis,25 butyrate-induced increase in GLP-1 leading
to improved glycemic status,26 reducing intestinal permeability and
improving epithelial barrier function,27 stimulating innate immune
response through membrane receptors in intestinal epithelial cells,28
stimulating TLRs with immune-regulatory effects on antiinflammatory
cytokines like IL-10 and transforming growth factor-β.29 So, it is cru-
cial to study the impact of supplementing probiotics on various
immune regulatory mechanisms operational in individuals with T1DM,
especially within the first few months of the disease's onset. We con-
ducted another study to assess the immune-regulatory markers of
T1DM following supplementation of probiotics and found a trend
towards improved immune-regulatory milieu (percentage of induced
T-regulatory cells, pancreatic antibody titers, and IL-10 in the blood)
in subjects receiving probiotics for 6 months (unpublished, partial data
presented at Virtual ISPAD 2020).30 So, we hypothesize that pro-
biotics could improve glycemic control by immune regulation of
beta-cell destruction by improving immune-regulatory milieu in the
pancreas. More such studies on immune-regulatory mechanisms
involved in the development of T1DM before and after supplementa-
tion of Probiotics (and Fasting & Stimulated C-peptide to study their
impact on beta-cell regeneration) are warranted.
Our study's strength is that it was a randomized, double-blind,
placebo-controlled study design, not only to determine the clinical
efficacy of the high strength probiotic preparation on glycemic
control but also to evaluate its safety profile in this age group. The
number of children completing the study was more than 93%. Fur-
ther, it is one of the first few studies assessing the efficacy of pro-
biotics in T1DM in a clinical setting, the previous studies being in
the NOD mice model.
Our study had a few limitations. The fecal microbiota analysis
could not be performed before and after the probiotic intervention to
study colonization and further justify the results. Since our study was
the first to assess the effects of high-strength multi-strain probiotic
preparation in children with T1DM, the study used a short treatment
regimen of 3 months. A brief intervention of 3 months, showing either
good or poor efficacy of the given intervention, may not be reliable as
glycemic control in these patients can be variable over shorter periods
due to factors other than the intervention being assessed. However,
we believe that the RCT design of our study should have taken care
of this limitation. Further, we did not analyze specific immune-
regulatory markers like IL10, GAD antibodies, and T-regulatory cells
to explore possible mechanisms responsible for probiotics' efficacy as
part of this study. However, we analyzed these immune-regulatory
markers before and after supplementation with probiotics as stated
above 30.
As our study results suggest a significant improvement in HbA1c
and reduction in insulin requirements in the probiotic group, we con-
clude that probiotics may have a supportive role in improving glyce-
mia among children with new-onset T1DM. However, more studies
with an extended intervention period are warranted to assess pro-
biotics' sustained effect over time. Further, the impact of probiotics
on markers of immune-regulation of beta-cell damage in T1DM needs
8 KUMAR ET AL.
to be studied to understand the mechanism of positive glycaemic
effects of probiotics.
ACKNOWLEDGMENTS
We appreciate the efforts of colleagues, fellows, and departmental
staff in the conduct of the study and are grateful for their assistance.
We acknowledge NextGen Pharma India Pvt. Ltd. for providing us
with supplies of investigational drug and placebo.
CONF LICT OF IN TE RE ST
None of the authors have any disclosures to report.
AUTHOR CONTRIBUTIONS
Rakesh Kumar, Sanjeev Kumar contributed towards the conception
and design of the study, recruited participants, monitored the con-
duct and progress of the research, interpreted the results, drafted,
and reviewed the manuscript. Latika Rohilla, Neenu Jacob, and
Jaivinder Yadav assisted with the study design, participant
enrolments, interpretation of results, and critical review of the
manuscript. Sanjeev Kumar, Rakesh Kumar, and Latika Rohilla were
responsible for management and coordination of the trial, monitor-
ing the study progress, data collection and its entry in database
files, and drafting of the manuscript. Naresh Sachdeva performed
laboratory investigations. Sanjeev Kumar, Rakesh Kumar, Jaivinder
Yadav, and Naresh Sachdeva analyzed and interpreted the data
and critically reviewed the manuscript. All the authors have read
and approved the final manuscript.
P EE R R EV I E W
The peer review history for this article is available at https://publons.
com/publon/10.1111/pedi.13244.
DATA AVAI LAB ILITY S TATEMENT
Data available on request from the authors
ORCID
Rakesh Kumar https://orcid.org/0000-0002-0039-2142
Latika Rohilla https://orcid.org/0000-0001-9803-779X
RE FE R ENC E S
1. Atkinson MA, Eisenbarth GS, Michels AW. Type 1 diabetes. Lancet.
2014;383:69-82.
2. Patterson CC, Dahlquist GG, Gyürüs E, Green A, Soltész G,
EURODIAB Study Group. Incidence trends for childhood type 1 diabe-
tes in Europe during 1989-2003 and predicted new cases 2005-20: a
multicentre prospective registration study. Lancet. 2009;373:2027-
2033.
3. DIAMOND Project Group. Incidence and trends of childhood type
1 diabetes worldwide 1990-1999. Diabet Med. 2006;23:857-866.
4. Das AK. Type 1 diabetes in India: overall insights. Indian J Endocrinol
Metab. 2015;19(Suppl 1):S31-S33.
5. Atkinson MA, Chervonsky A. Does the gut microbiota have a role in
type 1 diabetes? Early evidence from humans and animal models of
the disease. Diabetologia. 2012;55:2868-2877.
6. Vaarala O. The gut immune system and type 1 diabetes. Ann N Y Acad
Sci. 2002;958:39-46.
7. Luft FC. From toll-like receptors to the toll house of type 1 diabetes
mellitus. J Mol Med (Berl). 2010;88:1191-1194.
8. Lee AS, Ghoreishi M, Cheng WK, Chang TY, Zhang YQ, Dutz JP. Toll-
like receptor 7 stimulation promotes autoimmune diabetes in the
NOD mouse. Diabetologia. 2011;54:1407-1416.
9. Food and Agriculture Organization of the United Nations, World
Health Organization. Guidelines for the Evaluation of Probiotics in Food:
Report of a Joint FAO/WHO Working Group on Drafting Guidelines for
the Evaluation of Probiotics in Food. London, Ontario, Canada; WHO;
2002. http://www.who.int/foodsafety/fs_management/en/probiotic_
guidelines.pdf.
10. Murri M, Leiva I, Gomez-Zumaquero JM, et al. Gut microbiota in chil-
dren with type 1 diabetes differs from that in healthy children: a
case-control study. BMC Med. 2013;11:46.
11. Siljander H, Honkanen J, Knip M. Microbiome and type 1 diabetes.
EBioMedicine. 2019;46:512-521.
12. Stewart CJ, Ajami NJ, O'Brien JL, et al. Temporal development of the
gut microbiome in early childhood from the TEDDY study. Nature.
2018;562(7728):583-588.
13. Vatanen T, Franzosa EA, Schwager R, et al. The human gut micro-
biome in early-onset type 1 diabetes from the TEDDY study. Nature.
2018 Oct;562(7728):589-594.
14. Everard A, Cani PD. Diabetes, obesity and gut microbiota. Best Pract
Res Clin Gastroenterol. 2013;27:73-83.
15. Tremaroli V, Bäckhed F. Functional interactions between the gut
microbiota and host metabolism. Nature. 2012;489:242-249.
16. Balakumar M, Prabhu D, Sathish Kumar C, et al. Improvement in glu-
cose tolerance and insulin sensitivity by probiotic strains of Indian gut
origin in high-fat diet-fed C57BL/6J mice. Eur J Nutr. 2018 Feb;57(1):
279-295.
17. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group. Preferred
reporting items for systematic reviews and meta-analyses: the PRI-
SMA statement. Ann Intern Med. 2009;151:264-269.
18. Uusitalo U, Liu X, Yang J, et al. Probiotic use in infancy and islet auto-
immunity in the environmental determinants of diabetes in the young
(TEDDY) study. Diabetologia. 2014;57(suppl.1):S78-S79.
19. Ho J, Nicolucci AC, Virtanen H, et al. Effect of prebiotic on micro-
biota, intestinal permeability, and glycemic control in children with
type 1 diabetes. J Clin Endocrinol Metab. 2019;104(10):4427-4440.
20. Zare Javid A, Aminzadeh M, Haghighi-zadeh MH, Jamalvandi M. The
effects of Synbiotic supplementation on glycemic status, lipid profile,
and biomarkers of oxidative stress in type 1 diabetic patients. A
placebo-controlled, double-blind, randomized clinical trial. Diabetes
MetabSyndrObes Targets Ther. 2020;13:607-617.
21. Adly A, Ismail E, Salah N, Abd Elgawad M. The role of probiotics as an
adjuvant therapy for children with type 1 diabetes: effect on gylcemic
control and cytokine levels. Paper presented at 46th Annual Confer-
ence of ISPAD Virtual 2020; 2020 October 15–17 2020.
22. Eslamparast T, Zamani F, Hekmatdoost A, et al. Effects of symbi-
otic supplementation on insulin resistance in subjects with the
metabolic syndrome: a randomised, double-blind, placebo-
controlled pilot study. Br J Nutr. 2014;112(3):438-445. https://
doi.org/10.1017/S0007114514000919
23. Kooshki AA, Tofighiyan T, Rakhshani MH. Effects of synbiotics on
inflammatory markers in patients with type 2 diabetes mellitus. Glob J
Health Sci. 2015;7(7):1.
24. Boerner BP, Sarvetnick NE. Type 1 diabetes: role of intestinal micro-
biome in humans and mice. Ann N Y Acad Sci. 2011;1243:103-118.
25. Calcinaro F, Dionisi S, Marinaro M, et al. Oral probiotic administration
induces interleukin-10 production and prevents spontaneous autoim-
mune diabetes in the non-obese diabetic mouse. Diabetologia. 2005;
48:1565-1575.
KUMAR ET AL. 9

26. Yadav H, Lee JH, Lloyd J, Walter P, Rane SG. Beneficial metabolic ePoster presented at 46th Annual Conference of ISPAD Virtual 2020;
effects of a probiotic via butyrate-induced GLP-1 hormone secretion. 2020 October 15–17.
J Biol Chem. 2013;288:25088-25097.
27. Tian P, Li B, He C, et al. Antidiabetic (type 2) effects of lactobacillus
G15 and Q14 in rats through regulation of intestinal permeability and SUPPORTING INF ORMATION
microbiota. Food Funct. 2016;7:3789-3797. Additional supporting information may be found online in the
28. Kingma SD, Li N, Sun F, Valladares RB, Neu J, Lorca GL. Lactobacillus
Supporting Information section at the end of this article.
johnsonii N6.2 stimulates the innate immune response through toll-
like receptor 9 in Caco-2 cells and increases intestinal crypt Paneth
cell number in biobreeding diabetes-prone rats. J Nutr. 2011;141:
1023-1028. How to cite this article: Kumar S, Kumar R, Rohilla L, Jacob N,
29. Aumeunier A, Grela F, Ramadan A, et al. Systemic toll-like receptor Yadav J, Sachdeva N. A high potency multi-strain probiotic
stimulation suppresses experimental allergic asthma and autoimmune improves glycemic control in children with new-onset type 1
diabetes in NOD mice. PLoS One. 2010;5:e11484.
diabetes mellitus: A randomized, double-blind, and placebo-
30. Lokesh MN, Kumar R, Sachdeva N, Rawat A, Yadav J, Dayal D. Effect
of high strength oral probiotics supplementation on immune- controlled pilot study. Pediatr Diabetes. 2021;1-9. https://doi.
regulatory markers in children with new onset type one diabetes org/10.1111/pedi.13244
mellitus: a randomized, double-blind placebo control pilot trial.