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\begin{document}

\title{Alteration of gut microflora and role of gut dysbiosis in modulation of
gastrointestinal toxicity in pediatric cancer patients}



\author[1]{Debasish Sahoo}%
\author[1]{V. Deepak Bamola}%
\author[1]{Aditya Gupta}%
\author[1]{Jagdish Meena}%
\author[2]{Priyanka Naranje}%
\author[1]{Sadanand Dwivedi}%
\author[1]{Vineet Ahuja}%
\author[1]{Rama Chaudhry}%
\author[1]{Rachna Seth}%
\affil[1]{All India Institute of Medical Sciences}%
\affil[2]{AIIMS}%


\vspace{-1em}



  
  \date{June 12, 2024}


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\selectlanguage{english}
\begin{abstract}
Background Chemotherapy related mucosal toxicity is a major hindrance to
successful therapy in pediatric cancers. The role of gut dysbiosis in
modulation of chemotherapy related gastrointestinal toxicity is poorly
understood. Methods Pediatric cancer patients with neutropenia and
gastrointestinal symptoms were evaluated for neutropenic enterocolitis
(NEC) with CECT abdomen. Clinical features, fecal calprotectin and
microbiological data were analysed. Fecal Gut microbiota was evaluated
in children with NEC and compared with children where NEC was excluded
and healthy controls using conventional culture method. Results Of 590
children receiving chemotherapy during study period, 44 were diagnosed
with NEC. Significantly higher frequency of isolation of Bacteroides was
observed in children with NEC (42\%) as compared to non- NEC group
(14\%) and healthy controls (13\%). Isolation of Lactobacilli was
infrequent in NEC group (26\%) than non- NEC group (74\%) and healthy
controls (80\%). There was nonsignificant trend towards higher isolation
of Clostridium in children with NEC. Clostridiodes difficle or
Clostridium septicum were not identified in any group. Isolation of
other bacterial flora was similar in the sub groups. No significant
association of survival with gut dysbiosis could be established.
Isolation of Lactobacilli was associated with reduction in duration of
intravenous alimentation by 2.4 days, whereas isolation of Bacteroides
prolonged the requirement of bowel rest by 2.2 days. Conclusion Gut
dysbiosis was significantly higher in NEC group and associated with
higher morbidity suggesting its role in pathogenesis. This highlights
role of interventions towards gut dysbiosis like prebiotics and
probiotics in pediatric cancer patients.%
\end{abstract}%



\sloppy


\textbf{Manuscript title} : Alteration of gut microflora and role of gut
dysbiosis in modulation of gastrointestinal toxicity in pediatric cancer
patients

\textbf{Author affiliations}

\begin{enumerate}
\tightlist
\item
  Debasish Sahoo, DM, Pediatric Oncology Senior Resident Division of
  Pediatric Oncology Department of Pediatrics All India Institute of
  Medical Sciences, New Delhi, India debasish0712@gmail.com
\item
  V. Deepak Bamola, PhD Scientist Department of Microbiology All India
  Institute of Medical Sciences, New Delhi, India vdbamola@gmail.com
\item
  Aditya Kumar Gupta, MD
\end{enumerate}

Assistant Professor Division of Pediatric Oncology Department of
Pediatrics All India Institute of Medical Sciences, New Delhi, India
adivick@gmail.com

Jagdish Prasad Meena, MD Associate Professor

Division of Pediatric Oncology Department of Pediatrics All India
Institute of Medical Sciences, New Delhi, India drjpmeena@gmail.com

Sada Nand Dwivedi, PhD Professor Department of Biostatistics

All India Institute of Medical Sciences, New Delhi, India
dwivedi7@gmail.com

\begin{enumerate}
\tightlist
\item
  Priyanka Naranje, MD Associate Professor Department of Radiodiagnosis
  All India Institute of Medical Sciences, New Delhi, India
  Priyanka11sh@gmail.com
\item
  Vineet Ahuja, DM Professor Department of Gastroenterology All India
  Institute of Medical Sciences, New Delhi, India vineet.aiims@gmail.com
\item
  Rama Chaudhry, MD Professor and Head Department of Microbiology All
  India Institute of Medical Sciences, New Delhi, India
  drramach@gmail.com
\item
  Rachna Seth, MD Professor Division of Pediatric Oncology Department of
  Pediatrics All India Institute of Medical Sciences, New Delhi, India
  drrachnaseth1967@gmail.com
\end{enumerate}

\textbf{Address for correspondence}

\textbf{Corresponding author:} Dr Rachna Seth

\textbf{Postal Address:} Professor, Pediatric oncology,

Department of Pediatrics,

All India Institute of Medical Sciences (AIIMS), New Delhi, India,
110029

\textbf{Email:} drrachnaseth1967@gmail.com

\textbf{Phone:} 011- 26594345

91- 9971188687

\textbf{Word Counts}

For Abstract: 247

For Text: 3492

\textbf{Number of tables} : 5

\textbf{Number of figures:} 0

\textbf{Running title:} Gut dysbiosis in cancer chemotherapy

\textbf{Keywords:} Neutropenic enterocolitis, children, cancer, gut
microbiota, microflora, dysbiosis, mucositis

\textbf{Funding} : None

\textbf{Conflicts of interest:} None

\textbf{Abbreviations}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
NEC & Neutropenic enterocolitis\tabularnewline
\midrule
\endhead
CECT & Contrast enhanced computerised tomography\tabularnewline
ELISA & Enzyme linked immunosorbent assay\tabularnewline
HSCT & Hematopoietic stem cell transplant\tabularnewline
IBD & Inflammatory bowel disease\tabularnewline
IBS & Irritable bowel syndrome\tabularnewline
SCFA & Short chain fatty acid\tabularnewline
CMV & Cytomegalovirus\tabularnewline
PCR & Polymerase chain reaction\tabularnewline
RCM & Robertson cooked meat medium\tabularnewline
BHIBA & Brain heart infusion blood agar\tabularnewline
BBEA & Bacteroides bile esculin agar\tabularnewline
CCFA & Cefoxitin cycloserine fructose agar\tabularnewline
MRSA & deManRogosa agar\tabularnewline
AML & Acute myeloid leukemia\tabularnewline
ALL & Acute lymphoblastic leukemia\tabularnewline
NHL & Non Hodgkin lymphoma\tabularnewline
GVHD & Graft versus host disease\tabularnewline
COG & Children's Oncology Group\tabularnewline
\bottomrule
\end{longtable}

\textbf{Abstract}

\textbf{Background}

Chemotherapy related mucosal toxicity is a major hindrance to successful
therapy in pediatric cancers. The role of gut dysbiosis in modulation of
chemotherapy related gastrointestinal toxicity is poorly understood.

\textbf{Methods}

Pediatric cancer patients with neutropenia and gastrointestinal symptoms
were evaluated for neutropenic enterocolitis (NEC) with CECT abdomen.
Clinical features, fecal calprotectin and microbiological data were
analysed. Fecal Gut microbiota was evaluated in children with NEC and
compared with children where NEC was excluded and healthy controls using
conventional culture method.

\textbf{Results}

Of 590 children receiving chemotherapy during study period, 44 were
diagnosed with NEC. Significantly higher frequency of isolation of
Bacteroides was observed in children with NEC (42\%) as compared to non-
NEC group (14\%) and healthy controls (13\%). Isolation of Lactobacilli
was infrequent in NEC group (26\%) than non- NEC group (74\%) and
healthy controls (80\%). There was nonsignificant trend towards higher
isolation of \emph{Clostridium} in children with NEC.\emph{Clostridiodes
difficle} or \emph{Clostridium septicum} were not identified in any
group. Isolation of other bacterial flora was similar in the sub groups.

No significant association of survival with gut dysbiosis could be
established. Isolation of \emph{Lactobacilli} was associated with
reduction in duration of intravenous alimentation by 2.4 days, whereas
isolation of \emph{Bacteroides} prolonged the requirement of bowel rest
by 2.2 days.

\textbf{Conclusion}

Gut dysbiosis was significantly higher in NEC group and associated with
higher morbidity suggesting its role in pathogenesis. This highlights
role of interventions towards gut dysbiosis like prebiotics and
probiotics in pediatric cancer patients

\textbf{Introduction}

Neutropenic enterocolitis (NEC) is a life-threatening oncological
emergency observed in cancer patients during
chemotherapy.\textsuperscript{1} It has been synonymously termed
typhlitis, cecitis and ileo-caecal syndrome. The term typhlitis was
derived from the Greek word- \emph{Typhlon} , meaning caecum, indicating
the most common region of bowel involved in this
disease.\textsuperscript{2} Initially described as a triad of
neutropenia, pain abdomen and fever occurring in children with acute
leukemia, it has been described in adults as well as in other solid
malignancies and non-neoplastic conditions like aplastic anemia, post
hematopoietic stem cell transplantation (HSCT) and retroviral
infection.\textsuperscript{3,4}

The determinants of NEC are incompletely understood. Chemotherapy
related mucosal damage and neutropenia along with superadded infection
have been considered as the pathogenic mechanisms of NEC. The relative
avascularity and distensibility of certain segments like caecum
contribute to the pathogenesis. However, the role of gut dysbiosis in
etiopathogenesis of NEC is incompletely understood.

Human microbiome refers to the summation of all the microorganisms
residing in and on the human body. It is highly complex in composition
and diversity. The gut microbiota constitutes the largest microbiota in
the human body, accounting for about 70\% of microbial flora. Any
dysregulation in the interaction, quantity or quality of microbiota is
termed as dysbiosis. Qualitative alteration may take the form of loss of
alpha diversity (degree of microbial species diversity within a single
anatomical site) or beta diversity (degree of microbial diversity in the
same site between two individuals or groups).\textsuperscript{5}

Gut dysbiosis has been well described in diseases like inflammatory
bowel disease (IBD), irritable bowel syndrome (IBS) and\emph{Clostriodes
difficile} infection. However, the role of gut dysbiosis in pediatric
oncology has not been adequately explored. Gut dysbiosis has been
proposed to influence various aspects of cancer therapeutics:
therapeutic effect, toxicity including mucositis and even long-term
effects like obesity and neurocognitive functions.\textsuperscript{6}
Gut dysbiosis has also been linked to development of graft vs host
disease and increased non-relapse mortality during
HSCT.\textsuperscript{7,8} Conversely, gut dysbiosis has been noted in
relation to cancer chemotherapy, radiotherapy, immunotherapy and
antimicrobials: both prophylactic as well as therapeutic.

Gut dysbiosis has been proposed to play a key role in the pathogenesis
of gastrointestinal mucositis. Proposed mechanisms include modulation of
inflammatory and oxidative stress, production of protective short chain
fatty acids (SCFA), promotion of goblet cell function and epithelial
repair and promotion of local immunity.\textsuperscript{9} Gut dysbiosis
in response to chemotherapy, as noted in preclinical as well as human
studies, includes decrease in Shannon index, a marker of alpha
diversity, and alteration in the relative abundance of various species.
Decrease in the abundance of \emph{Firmicutes}
including\emph{Lactobacilli} and increase in\emph{Bacteroides}
\textsuperscript{10-12} has been noted in few studies, whereas the
converse has been observed in others.\textsuperscript{13,14}

With the above gaps in knowledge, a prospective observational study was
planned to assess the gut dysbiosis in pediatric cancer patients with
NEC and the prognostic importance of the same.

\textbf{Methodology}

It was a descriptive study conducted in a tertiary health care centre in
India between January 2019 to December 2020. Pediatric cancer patients
aged 6 months to 18 years were included in the study. Ethical clearance
from the IRB was taken. Informed consent was taken. All the children
receiving chemotherapy during the study period formed the denominator of
the study

\textbf{Screening and evaluation}

Children presenting with neutropenia and any gastrointestinal symptoms
(pain abdomen, vomiting, abdominal distension, diarrhoea, or
haematochezia) were evaluated. Conditions like acute gastritis, acute
pancreatitis and acute appendicitis were excluded from the study by
appropriate clinical evaluation and investigations. After excluding the
above patients, patients were enrolled in the study.

The enrolled patients underwent CECT abdomen. Based on the physical
findings and findings of CECT abdomen, they were classified into:
Definite NEC, Probable NEC and NEC excluded. Patients with definite and
probable NEC underwent further laboratory evaluation, which included
serial blood counts, bacterial and fungal work up for isolation of
offending organism, stool work up, blood CMV PCR and fecal calprotectin.
Fecal calprotectin was analysed by sandwich ELISA method using
commercially available kits.

Children diagnosed as NEC were admitted and given standard care
according to institutional protocol, which included intravenous
antimicrobials, bowel rest, intravenous fluids, correction of
electrolyte imbalances, transfusion support and parenteral nutrition.
Use of growth factors was decided based on unit protocol that was guided
by clinical picture, underlying malignancy and phase of chemotherapy.
Surgical intervention was considered in case of complications like
perforation, uncontrolled haemorrhage and uncontrolled sepsis despite
appropriate intravenous antimicrobials. They were followed up till
discharge. The outcome variables including death or discharge, duration
of hospital stay, duration of bowel rest, requirement of vasoactive
agents and need for mechanical ventilation were recorded.

Faecal microflora was analysed in the enrolled patients- those with
diagnosis of NEC and NEC excluded, as well as in healthy controls. The
healthy controls included siblings of children with cancer and were
required to be asymptomatic and not having received any antibiotics in
the preceding one month.

\textbf{Definitions}

\textbf{Neutropenic enterocolitis} :

There is no standardized definition for neutropenic enterocolitis, with
wide variability in the definition across the studies. For purpose of
current study, all patients on cancer chemotherapy who were neutropenic
and had gastrointestinal symptoms were eligible for evaluation. Patients
were classified into three groups i). NEC excluded ii.) probable NEC and
iii.) definite NEC. This was done after other causes like pancreatitis,
gastritis appendicitis and ileus due to metabolic derangements like
hypokalemia were ruled out and there was persistence of symptoms beyond
48 hours.

\textbf{NEC excluded:} Patients having neutropenia along with any of the
gastrointestinal symptoms but no abnormal findings on physical
examination (tenderness, rebound tenderness, rigidity) and no
radiological abnormality.

\textbf{Probable NEC:} Patients having neutropenia and gastrointestinal
symptoms with abnormal physical findings, but no radiological
abnormality.

\textbf{Definite NEC} : Patients having neutropenia and gastrointestinal
symptoms, with abnormal findings on physical examination and positive
radiological criteria.

Both probable \& definite NEC were considered as NEC for this study.

\textbf{Radiological criteria} : Patients with suspected NEC satisfying
entry criteria underwent CECT abdomen. Findings (one/more) suggestive of
NEC served as radiological criteria- bowel wall thickening
\textgreater{} 3 mm, abnormal bowel dilatation, pneumatosis intestinalis
or free intraabdominal gas.

\textbf{Statistical analysis}

Descriptive statistics were used to analyse the demographic and clinical
characteristics of the patients. Mean (SD) and median (IQR) were
performed for continuous variables. Comparison between categorical
variables was performed using the Pearson Chi-square test or Fisher's
exact test and between continuous variables using the t-test or Wilcoxon
rank-sum/ Mann-Whitney test. Independent association between variables
and outcome was assessed by logistic regression. The level of
significance was set at a p-value of \textless{}0.05. The analyses were
performed using Stata software version 20 (STATA, College Station, TX).

\textbf{Evaluation of gut microflora}

Conventional stools culture methods were used to evaluate the
predominant bacterial flora in the stool samples. Fresh stool samples
were immediately transported to the laboratory for evaluation of
microflora. Various media were used to isolate and identify both aerobic
and anaerobic microflora. MacConkey agar and Blood Agar were used for
isolation of aerobic bacteria, whereas Robertson Cooked Meat medium
(RCM), brain heart infusion blood agar (BHIBA), Bacteriodes Bile Esculin
agar (BBEA), cefoxitin-cycloserine fructose agar (CCFA) and deManRogosa
agar (MRSA) were used for anaerobic bacteria. MacConkey agar and blood
agar are incubated aerobically, the other media used for anaerobic
bacteria are incubated under anaerobic conditions. BHIBA is a general
enriched medium that supports the growth a wide range of anaerobic
bacteria. CCFA medium is a selective medium for \emph{Clostridiodes
difficile} , with the addition of antibiotics- cycloserine and
cefoxitin, which inhibit the growth of other aerobic and anaerobic
bacteria. To detect spore bearing \emph{Clostridia} spp. the stool
samples were subjected to alcohol shock treatment before culturing on
CCFA, wherein about O.5 ml. of the sample was first treated with equal
amounts of 100\% ethanol for 60 min. at 37 \textsuperscript{0}C (alcohol
treatment) prior to plating. BBE medium is used for isolation of
\emph{Bacteroides}species, the medium is rendered selective by addition
of oxgall and gentamicin at a concentration less than 80\selectlanguage{greek}µ\selectlanguage{english}g/mL, which
inhibit growth of other bacteria. MRS medium is a selective medium
for\emph{Lactobacillus} species. Further species identification was done
using matrix assisted laser desorption ionization-time of flight mass
spectrometry (MALDI-TOF MS bioMerieux).

\textbf{Results}

A total of 590 children with cancer received chemotherapy during the
study period of January 2019 to December 2020. There were 224 children
who presented with neutropenia with presence of gastrointestinal
symptoms and were screened for NEC. Evidence of acute pancreatitis based
on biochemical and imaging findings was detected in three children and
were excluded. A diagnosis of acute gastritis, made after therapeutic
trial of proton pump inhibitors, or rapid resolution of symptoms within
48 hours was observed in 149 children. The rest 72 episodes were
evaluated for NEC and enrolled into the study.

They were evaluated for NEC based on the study protocol. Based on the
physical findings and radiological investigations, they were classified
into probable and definite NEC and NEC excluded. NEC was excluded in 24
cases and the diagnosis was considered in 48 episodes, 36 had definite
NEC according to the definition and 12 had probable NEC. These 48
episodes were observed in 44 children, with 4 children developing
recurrent episodes.

\textbf{Baseline characteristics}

Of the 48 cases with NEC, there were 25 males and 23 females. Most of
them belonged to age group 3-5 years (42\%) followed by adolescent
children (25\%). Acute myeloid leukemia (AML) was the most common
underlying diagnosis, accounting for 31\% of the episodes. It was
followed by acute lymphoblastic leukemia (ALL) (29\%), non-Hodgkin
lymphoma (NHL) (13\%) and relapsed ALL (8\%). The proportion of children
developing NEC was the highest in NHL (40\%), followed by AML (32\%) and
relapsed ALL (20\%). Fifty percent of children received anthracyclines
and etoposide prior to development of NEC. Cytarabine based chemotherapy
was administered prior to 45\% of episodes. Twenty percent of the
children received methotrexate-based chemotherapy. Steroids were
administered in 35\% of the cases. Alkylating agents were administered
in 29\% of the cases. In 26 of 48 episodes (54\%), children developed
NEC following the first cycle of chemotherapy.

\textbf{Gut microflora}

Gut microflora was available in 38 children with NEC, 21 children where
NEC was excluded and 30 healthy controls. \emph{Bacteroides} were
isolated with a significantly higher frequency in the NEC group than
non- NEC group and healthy controls, with isolation rates of 42\%, 14\%
and 3\% respectively, which was statistically significant. Similarly,
there was a trend towards higher frequency of isolation
of\emph{Clostridium} in children with NEC; it could be isolated in 37\%
of children with NEC and 19\% in children without NEC as compared to
13\% in healthy controls, however this was not statistically
significant. Interestingly, \emph{Clostridiodes difficile} was not
isolated in any of the samples. \emph{Clostridium septicum,} a
well-recognised pathogenic species, was not grown in any of the groups.
The `healthy bacteria',\emph{Lactobacillus} species were isolated in
26\% of children in NEC group as compared to 74\% and 80\% in non-NEC
group and healthy controls respectively (p \textless{} 0.001). The other
`healthy bacteria'\emph{Bifidobacterium} was not isolated in any of the
samples. The proportion of other anaerobic flora like \emph{Veillonella}
and aerobic flora like \emph{E. coli, Klebsiella, Enterococcus}
and\emph{Citrobacter} was not significantly different in any of the
groups. (Table 1)

\textbf{Prognostic significance of gut microflora}

A mortality rate of 23\% (n = 11) was observed in the study cohort.
Higher isolation rates of \emph{Clostridium} and \emph{Bacteroides} were
observed in children who didn't survive the illness; however, this
difference was not statistically significant. The isolation of other
bacterial flora was similar in the non survivors and survivors. (Table
2)

Presence of gut dysbiosis was found to be associated with prolonged
requirement of bowel rest and intravenous alimentation. Isolation
of\emph{Lactobacilli} was associated with reduction in duration of
intravenous alimentation by 3.8 days by univariate analysis, whereas the
isolation of \emph{Bacteroides} prolonged the requirement of bowel rest
by 2.3 days. This association retained statistical significance on
multivariate analysis, with adjusted coefficient of -2.4 and 2.2
respectively. No association was noted between gut dysbiosis and other
outcome parameters like duration of hospitalization and requirement of
vasoactive support and mechanical ventilation.

Faecal calprotectin was found to be significantly elevated in patients
where \emph{Enterococcus} was not isolated as compared to those who
showed growth of \emph{Enterococcus} (151\selectlanguage{greek}µ\selectlanguage{english}g/g vs 64\selectlanguage{greek}µ\selectlanguage{english}g/g, p = 0.005).
However, isolation of no other bacteria showed any association with
faecal calprotectin. (Table 3) There was no significant association
between gut microflora with serum procalcitonin or degree of
neutropenia.

\textbf{Discussion}

The concept of gut microbiota is highly complex and is incompletely
understood. Interaction of the gut microbiome with the intestinal mucosa
plays a key role in the maintenance of mucosal integrity. The proposed
mechanisms include stimulation of local mucosal immunity, production of
antimicrobial substances like bacteriocin that inhibit growth of
pathogenic microorganisms and sustenance of the intestinal mucosal cells
by production of vitamins and short chain fatty
acids.\textsuperscript{15-18} Gut dysbiosis may be affected due to
several causes like geography, age, diet, antibiotics exposure and toxic
exposure. Gut dysbiosis has been studied in several disease states like
IBD, IBS, \emph{Clostridiodes difficile} infection and carcinoma
colon.\textsuperscript{18-21} In fact, few therapeutic approaches target
the gut dysbiosis with interventions like fecal microbiota
transplant.\textsuperscript{22,23}

Several preclinical studies done in murine models have demonstrated the
alteration of gut microbiota in response to cancer chemotherapy. Most of
them have demonstrated a reduction in alpha diversity and reduction
in\emph{Lactobacilli} , while there are conflicting results
regarding\emph{Bacteroides} and \emph{Clostridium}
.\textsuperscript{24-28} Of note, Bultzingslowen et al found increase in
gut translocation of gram-negative bacteria to mesenteric lymph nodes
after chemotherapy, which was ameliorated with addition of
\emph{Lactobacillus plantarum 299v} .\textsuperscript{29} There is scant
literature regarding role of gut dysbiosis in setting of pediatric
cancer, more so in mucositis and neutropenic enterocolitis. Various
aspects of therapy of cancer including chemotherapy, radiotherapy,
immunotherapy, infections and use of antibiotics have been known to
alter the gut microbiota.\textsuperscript{30-33}. The presence of gut
dysbiosis also has bearing on various aspects of cancer therapeutics-
therapeutic effect\textsuperscript{34-36}, chemotherapy related toxicity
including mucositis\textsuperscript{37-39} and even long-term effects.
In setting of HSCT, it also has been proposed to influence the incidence
of bacteraemia\textsuperscript{40,41} and development of graft vs host
disease (GVHD)\textsuperscript{7,8} and non-relapse
mortality\textsuperscript{42}.

In the current study, gut dysbiosis was observed in form of higher
isolation of \emph{Bacteroides} and lower isolation
of\emph{Lactobacilli} in patients with NEC as compared to healthy
controls. A trend towards higher isolation of \emph{Clostridium} was
also observed in patients with NEC, however, it was not statistically
significant. Various studies have uniformly noted a decrease in alpha
diversity, as measured by Shannon index, as a marker of gut
dysbiosis.\textsuperscript{5} Most of them also reported decrease in
isolation of \emph{Lactobacillus} and \emph{Bifidobacterium} as a part
of gut dysbiosis. However, there are conflicting reports
on\emph{Bacteroides} and other \emph{Firmicutes} as markers for
dysbiosis of faecal microbiota. A systematic review on the alteration of
gut microbiota in cancer chemotherapy related mucositis found a
significant reduction of alpha diversity as measured by Shannon index
and reduction in \emph{Lactobacilli, Bifidobacterium, and Clostridium}
species (\emph{Firmicutes} ), coupled with increase in
\emph{Bacteroides}species.\textsuperscript{5,49} (Table 4) Van Vliet et
al studied the gut dysbiosis in nine pediatric AML patients prior to
initiation of chemotherapy and after each cycle of
chemotherapy.\textsuperscript{37}There was decrease in alpha diversity
as measured by Shannon index, even prior to initiation of chemotherapy,
and after each cycle of chemotherapy. There was a decrease in number of
all genera after chemotherapy except \emph{Enterococcus} . Huang et al
found a similar trend of lower bacterial counts in pediatric patients
with ALL, which was exacerbated after administration of high dose
methotrexate.\textsuperscript{43} There were also lower counts
of\emph{Bifidobacterium, Lactobacillus} and \emph{Escherichia coli} in
the patients as compared to controls. Administration of chemotherapy
resulted in further lowering of counts of the above bacteria. Stringer
et al studied the gut microbiota in 16 adult patients on various
chemotherapy, they were found to have decrease in \emph{Bacteroides,
Bifidobacterium} and \emph{Lactobacillus} along with increase in\emph{E.
coli} and \emph{Staphylococci} as compared to healthy
controls.\textsuperscript{44} The above studies observed decrease
in\emph{Bacteroides} along with \emph{Bifidobacterium}
and\emph{Lactobacillus} as features of gut dysbiosis. Contrastingly,
other studies by Rajagopala et al\textsuperscript{45}, Montassier et
al\textsuperscript{46}, Zwielehner et al\textsuperscript{47} and Chua LL
et al\textsuperscript{48} have observed increase in
\emph{Bacteroides}and fall in \emph{Firmicutes} (\emph{Clostridium}
and\emph{Lactobacillus)} as markers of gut dysbiosis. Rajagopala et al
found a lower alpha diversity in pediatric cancer patients,
lower\emph{Lachnospaerae} and abundance of \emph{Bacteroides} as
compared to controls. Chua et al observed gut dysbiosis in pediatric ALL
patients in form of fall in Shannon index, increase in
\emph{Bacteroides} and decrease in \emph{Firmicutes} . A similar finding
was observed by Montassier et al in adult patients undergoing HSCT.
Zwielehner et al also found a decrease in alpha diversity and increase
in\emph{Bacteroides} and \emph{Clostridium} cluster IV with decrease
in\emph{Bifidobacterium} and \emph{Clostridium} cluster IVa. This
difference in features of gut dysbiosis may be explained by racial,
geographical, and dietary differences and difference in age group.

The relative abundance of each bacteria did not have any significant
association with mortality, which may be explained by the small sample
size in the non survivors. However, isolation of \emph{Bacteroides} and
absence of \emph{Lactobacilli} did have an overwhelming impact on
requirement of prolonged intravenous alimentation. Isolation
of\emph{Bacteroides} increased the requirement of bowel rest by 2.2 days
and presence of \emph{Lactobacilli} tended to decrease the requirement
of intravenous alimentation by 2.4 days. This observation points to a
strong protective effect of flora like \emph{Lactobacillus} and
disruptive effect of \emph{Bacteroides} on the integrity of
gastrointestinal tract. This observation supports the hypothesis and
mechanism of role of gut microbiota as proposed by van Vliet et
al\textsuperscript{9}.

Various mechanisms have been proposed to explain the role of `healthy'
gut microbiota in modulating the chemotherapy related gastrointestinal
toxicity. Gut microbiota and their secretions have been proposed to
decrease the inflammation by decreasing NF\selectlanguage{greek}κ\selectlanguage{english}B activation and thus
downregulating TLR and IL-6, while inducing anti-inflammatory agents
like IL-10. Short chain fatty acids (SCFA) like butyrate secreted by the
microbiota may also ameliorate the inflammatory and oxidative damage in
the setting of infection. Microbiota may decrease the villous atrophy
and epithelial cell loss induced by chemotherapy. Certain bacteria
like\emph{Lactobacillus rhamnosus GG} and \emph{Lactobacillus
acidophilus}upregulate the expression of \emph{MUC- 2} and \emph{MUC- 3}
genes, which promotes goblet cell repair and mucin production. They also
promote the repair of the mucosa though protective effect of SCFAs.
Finally, they contribute to local immunity through secretion of
protective molecules like C type lectins, secretory IgA and
cathelicidins, which have antibacterial activity and prevent gut
translocation of bacteria. Findings of the study strongly support the
role of gut dysbiosis in the pathogenesis of neutropenic enterocolitis
and protective role of `healthy flora'.

This observation supports the hypothesis that use of appropriate
prebiotics or probiotics may improve the gut dysbiosis in pediatric
cancer patients and help improve the treatment related morbidity and
mortality. There are only limited studies evaluating role of prebiotics
or probiotics in pediatric cancers. (Table 5) Ekart et al observed a
reduction in incidence of febrile neutropenia with minimal increase in
adverse effects in children who were administered
\emph{Lactobacilli}with prophylactic cotrimoxazole as compared with
others receiving a cocktail of prophylactic
antibiotics.\textsuperscript{50} Zheng et al observed a favourable
alteration in gut microbiota in patients administered
fructooligosachharide, which served as prebiotics.\textsuperscript{51}
Wade et al found a reduction in incidence of fever and antibiotic
exposure along with increase in stool SCFA in children who received
probiotics.\textsuperscript{52} Another study found a reduction in grade
3-4 diarrhoea and mucositis in adults receiving prebiotics during HSCT,
though it did not translate into a benefit in
survival.\textsuperscript{53} A recent RCT in adults evaluating role of
probiotic- \emph{Lactobacillus rhamnosus} for prevention of GVHD was
prematurely terminated owing to lack of efficacy. However, the
intervention was started only on day 14 of transplant. A pilot study in
pediatric patients undergoing HSCT to evaluate safety of probiotic-
\emph{Lactobacillus plantarum} did not show any increase in side
effects.\textsuperscript{55} Children's Oncology Group (COG) ACCL 1663
trial is currently underway to study the efficacy and safety of the
probiotics in children undergoing HSCT.

\textbf{Limitations and strengths of the study}

Analysis of gut microbiota by NGS couldn't be done in view of financial
constraints, hence analysis of gut microflora was done by conventional
culture method. While it showed the growth of the predominant
microbiota, bacterial flora present in smaller number were not detected.
This also precluded as estimation of Shannon index.

This was the first prospective study on NEC during its commencement. A
strict and robust diagnostic criterion was used for the diagnosis of
NEC. Role of the biomarker- faecal calprotectin was evaluated in this
setting for the first time. There are no studies which studied the role
of gut microbiota in pediatric cancer patients with neutropenic
enterocolitis. The presence of gut dysbiosis seen in the study may
reflect a pathogenic mechanism of neutropenic enterocolitis. This gives
basis for further studies on gut microbiota in the field of pediatric
oncology and possible role of interventions like prebiotics and
probiotics.

\textbf{Conflict of interest:} None

\textbf{Funding:} None

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\end{enumerate}

\par\null

\textbf{TABLE 1: Faecal microflora in children with and without
neutropenic enterocolitis and healthy controls}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{S. No}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{Microorganism}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{NEC} \textbf{n = 38}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{NEC excluded} \textbf{n = 21}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{Healthy controls} \textbf{n = 30}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\textbf{p value}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Clostridium}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
14 (36.8)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 (19.0)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 (13.3)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.06\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Bacteroides}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
16 (42.1)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3 (14.2)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 (13.3)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.01\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Veillonella}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 (10.5)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1 (4.7)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1 (3.3)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.46\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Lactobacilli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
5 (26.3)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
15 (73.9)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
24 (80)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textless{}0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{E. coli}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
30(78.9)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
16 (76.1)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
26 (86.6)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.59\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Enterococcus}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
8 (21.0)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3 (14.2)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
6 (20)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.80\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Klebsiella}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4 (10.5)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3 (14.2)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
6 (20)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.54\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\emph{Citrobacter}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3 (7.8)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2 (9.5)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1 (3.3)\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.63\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 2: Association of faecal microflora with survival in
patients with neutropenic enterocolitis}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{S. No}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Faecal microbiota}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Survivors, n = 29} \textbf{n (\%)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Non survivors, n = 9} \textbf{n (\%)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{p value}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Clostridium\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
10 (34.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (44.4)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.43\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Bacteroides\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12 (41.3)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5 (55.5)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.35\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~3\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Veillonella\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
3 (10.3)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (11.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.67\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Lactobacilli\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (13.8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (11.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.66\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~5\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
E coli\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
24 (82.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7 (77.7)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.53\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~6\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Enterococcus\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
7 (24.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (22.2)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.64\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
~7\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Klebsiella\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4 (13.8)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0 (0)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.32\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Citrobacter\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
2 (6.9)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1 (11.1)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.56\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 3: Association of specific genera in faecal microflora
with faecal calprotectin,}

\textbf{n = 38}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{S. No} & \textbf{Faecal microbiota} & \selectlanguage{greek}\textbf{Faecal
calprotectin in specific genera (µg/g)}\selectlanguage{english} & \selectlanguage{greek}\textbf{Faecal calprotectin in
specific genera (µg/g)}\selectlanguage{english} & \textbf{p value}\tabularnewline
\midrule
\endhead
~1 & Clostridium & 137 (25-359) & 126 (46-380) & 0.44\tabularnewline
~2 & Bacteroides & 150 (25-359) & 115 (29-380) & 0.69\tabularnewline
~3 & Veillonella & 143 (78-278) & 129 (25-380) & 0.58\tabularnewline
~4 & Lactobacilli & 81 (36-126) & 138 (25-380) & 0.42\tabularnewline
~5 & E coli & 142 (25-380) & 79 (49-126) & 0.27\tabularnewline
~6 & Enterococcus & 64 (25-126) & 151 (29-380) & 0.005\tabularnewline
~7 & Klebsiella & 165 (79-278) & 127 (25-380) & 0.37\tabularnewline
8 & Citrobacter & 138 (29-286) & 130 (25-380) & 0.91\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 4: Alteration in gut microbiota with chemotherapy or
infections}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{S. No}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Study}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Population}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Sample size}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Technique}\strut
\end{minipage} & \begin{minipage}[b]{0.14\columnwidth}\raggedright\strut
\textbf{Results}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
1.\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Van Vliet et al\textsuperscript{37}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Pediatric, AML\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
9\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Decrease in Shannon index even prior to initiation of chemotherapy
Decrease in all genus with chemotherapy \emph{(Clostridium, Bacteroides,
Bifidobacterium)} Increase in \emph{Enterococcus} Partial recovery
during subsequent cycle of chemotherapy\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
2.\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Huang et al\textsuperscript{43}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Pediatric, ALL\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
36 patients 36 controls\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Lower total bacterial counts, lower \emph{Bifidobacteria, Lactobacilli}
and \emph{E. coli} than healthy controls prior to chemotherapy Further
fall in bacterial counts, \emph{Bifidobacteria, Lactobacilli} and
\emph{E. coli} after high dose methotrexate\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
3\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Stringer et al\textsuperscript{44}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Adults\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
16 patients\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Decrease in \emph{Bacteroides, Lactobacilli} and \emph{Bifidobacterium}
Increase in \emph{E. coli} and \emph{Staphylococci}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
4.\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Rajagopala et al\textsuperscript{45}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Children and young adults\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
28 patients 23 healthy controls\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Lower Shannon index in patients Lower \emph{Lachnospaerae}, higher
\emph{Bacteroides} than healthy controls No change in Shannon index post
chemotherapy, but increased subsequently\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
5.\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Montassier et al\textsuperscript{46}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Adults with NHL for HSCT\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
8 patients\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Decrease in Shannon index after starting chemotherapy Increase in
\emph{Bacteroides}, decrease in \emph{Fermecutes} and
\emph{Bifidobacterium}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Zwielehner et al\textsuperscript{47}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Adults\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
17 patients\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Decrease in total bacterial count and diversity after starting
chemotherapy Increase in \emph{Bacteroides} and \emph{Clostridium}
cluster IV Decrease in \emph{Bifidobacterium} and \emph{Clostridium}
cluster IVa\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
7\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Bamole VD et al1\textsuperscript{8}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Adult, Ca colon\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
8 patients, 16 controls\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Culture and PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Higher Bacteroides: Firmicutes ratio in patients Lactobacillus not grown
in any patient, 57\% in controls Dietary variation observed\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Chua LL et al\textsuperscript{48}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Pediatric; ALL\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
7 patients\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
PCR\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Decrease in alpha diversity even prior to initiation of chemotherapy
Relative decrease in firmicutes and abundance of Bacteroides prior to
therapy The difference diminished after completion of therapy, but
didn't normalise\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Index study\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Pediatric\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
-NEC: 38 -Non-NEC: 21 -Healthy controls: 30\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Culture methods\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Increase in \emph{Bacteroides} and decrease in \emph{Lactobacilli} in
NEC group Trend towards higher isolation of Clostridium in NEC
group\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{TABLE 5: Studies targeting gut dysbiosis in cancer patients}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{S. No}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Study}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Sample size}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Intervention}\strut
\end{minipage} & \begin{minipage}[b]{0.17\columnwidth}\raggedright\strut
\textbf{Result}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
1.\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Ekert et al\textsuperscript{50}, Australia, 1980\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
68 children with cancer\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Arm I- Lactobacilli + Cotrimoxazole Arm II- Framycetin + Nystatin +
Metronidazole\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Arm I showed lower incidence of febrile neutropenia Minimal side
effects, well tolerated\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
2.\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Zheng et al\textsuperscript{51}, China, 2006\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
67 pediatric cancer patients\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Intervention (32): Prebiotic- Fructooligosachharide Control: 35\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Higher lactobacilli count in intervention group than control Trend
towards higher Bifidobacterium in intervention group (Not
significant)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
3.\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Wada et al\textsuperscript{52}, Japan, 2010\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
42 pediatric cancer patients\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Intervention (19): Probiotic-\emph{Bifidobacterium breve} strain Yakult
Control: 23\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Lower incidence of fever and antibiotic exposure in intervention group
Lower count of enterococci in intervention group Higher SCFA in
intervention group\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
4.\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Iyama et al\textsuperscript{53}, Japan 2014\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
44 adult cancer patients undergoing HSCT\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Intervention (22): Prebiotic combination- Glutamine, fiber and
oligosachharide Control: 22\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Significantly lower incidence and duration of grade 3-4 diarrhoea and
mucositis Decreased weight loss and requirement of parenteral nutrition
Trend towards lower gut bacterial translocation in intervention group No
difference in OS, GVHD rates, though some survival benefit at day
100\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
5\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Gorshein et al\textsuperscript{54}, USA, 2017\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
31 adult cancer patients undergoing HSCT\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Intervention (20): Probiotic Lactobacillus rhamnosus GG started post
engraftment Control: 10\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
No reduction in TRM, rates of acute or chronic GVHD No difference in
microbiome as compared to controls Trial terminated early due to lack of
efficacy\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
6\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Ladas et al\textsuperscript{55}, USA, 2016\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
30 children undergoing HSCT, pilot study\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Probiotic \emph{Lactobacillus plantarum} started from day -8 of
transplant\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
Feasible Safe, no increase in toxicity\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

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