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Methodologies

omnia Amir
 1.

Analysis of viral diversity in stool samples from

infants and children with acute gastroenteritis
in Kuwait using Metagenomics approach
Hawraa Adel Mohammad, Nada Mohammed Madi and Widad Al-Nakib

https://doi.org/10.1186/s12985-020-1287-5
 1.

Analysis of viral diversity in stool samples from

infants and children with acute gastroenteritis
in Kuwait using Metagenomics approach
Hawraa Adel Mohammad, Nada Mohammed Madi and Widad Al-Nakib

https://doi.org/10.1186/s12985-020-1287-5
 Gastroenteritis is the second leading cause of
death among infants and children worldwide. It is
responsible for approximately three million deaths
each year, causing high morbidity and mortality
rate globally.

Although at least 25 different bacteria and

protozoa can cause diarrhoea, more than 75% of
cases found to be caused by viruses. Viruses that
can cause gastroenteritis include rotavirus,
norovirus, enteric adenovirus, human astrovirus,
and Sapporo virus. Currently, the diagnostic
methods used to detect viruses in stool samples
are sequence-dependent molecular amplification
techniques such as PCR, which cannot identify a
pool of viruses and completely new viruses in
clinical samples
 Methodology

Eighty-four infants and children with signs and
symptoms of gastroenteritis, including diarrhoea,
vomiting, fever, and abdominal pain from January
to December 2017 were enrolled in this study. The
patients aged between 1 and 10 years old (median
age = 2 years old) who attended Al-Amiri and
Mubarak Al-Kabeer Hospitals in Kuwait
The collected samples from patients and healthy
children were processed at the Virology Unit and
Research Core Facility/OMICS Research Unit,
Health Sciences Centre, Kuwait University, for the
presence of viruses causing gastroenteritis by
metagenomics approach using NGS and the
commercial multiplex Real-Time PCR assay.

Nucleic
acids The collected stool samples were re-suspended in
extraction phosphate buffer saline, incubated for 1 h at 4 °C
and centrifuged at 6000 Xg (5530 rpm) for 5 min at
4 °C to enrich the viruses. Total nucleic acids were
extracted from stool samples using automated
MagNA Pure LC system, according to the
manufacturer’s instructions.
 Next-generation sequencing and metagenomics analysis

The extracted nucleic acids (RNA and DNA) were
processed for metagenomics analysis using the
Illumina MiSeq platform for NGS according to
standard procedures. Briefly, the genomic host DNA
in the extracted nucleic acids was removed using
Ambion DNA-free following the manufacturer’s
instructions.
Quantification of cDNA was performed using
QubitR Fluorometer and Qubit™ dsDNA BR Assay
Kit (Invitrogen, California, USA) following the
manufacturer’s instructions and one ng of cDNA
was used for library preparation. DNA libraries
were prepared using Illumina TruSeq DNA Library
Preparation Kit V2 (Illumina San Diego, CA, USA).

The pooled DNA libraries were sequenced using

the Illumina MiSeq instrument at Research Core
Facility/OMICS Research Unit, Health Science
Centre, Kuwait University, to generate 150-bp
paired-end reads.
 Bioinformatics Step
After sequencing by Miseq sequencer, fastq sequence files
were checked for quality using Fastqc and low quality
ends, below 20 and above 240, were trimmed using
FASTX-Toolkit. The genome sequence of human, viruses,
and bacteria was downloaded from NCBI (National Center
for Biotechnology Information) RefSeq, and a custom
database was created using build option in Kraken
software.
Each paired-end fastq file was mapped to the
database using Kraken to assign taxonomic labels
to the sequences. For a detailed analysis of the
reads, fastq files were also mapped to the
database using BWA-MEM (Burrows-Wheeler
Alignment mem option) and sam files were
obtained for the alignment and were filtered for
MAPQ score 0.

Samtools flagstat was used for the information

about the percentage of reads aligned to each
database. The sam files generated from the BWA
program was analyzed by bbmap for detailed
analysis. Raw reads data with high-quality reads
were archived at NCBI as sequence read archive
(SRA) with BioProject accession number:
PRJNA587350.
 Multiplex real-time PCR assay Step :
New nucleic acid extractions from each sample
were prepared and used for multiplex Real-
Time PCR. Fast Track Diagnostic Kit (Fast-Track
Diagnostic, Luxemburg, Germany) was used to
detect viruses causing AGE according to the
manufacturer’s instructions.
Statistical
analysis
The assay is a routine diagnostic test which is
performed at the Virology Unit, Mubarak Al-Kabeer
Hospital, Kuwait, for the detection of viruses that
cause gastroenteritis including norovirus genotype
1 and 2; astrovirus; rotavirus; adenovirus; and
sapovirus. All runs were performed using the
LightCycler®480 instrument II (Roche Diagnostic,
Mannheim, Germany).
The data were analysed using computer software
“Statistical Package for Social Sciences”, SPSS version
25.0 (IBM Corp, Armonk, NY). The descriptive statistics
were presented as frequencies and percentages.
Cohen’s Kappa statistics (k) was applied to find the
agreement by both metagenomics and multiplex Real-
Time PCR assays. Two-tailed probability value P < 0.05
was considered statistically significant.
 Results
Metagenomics analysis revealed an average of 280,768 reads in which 5% of the reads
were derived from viruses. The analysis of viral sequences verified that single infection
of human adenovirus was the leading cause of gastroenteritis among infants and
children, which was detected in 23.2% of the patients, followed by a mixed infection of
human adenovirus and other viruses, which was detected in 20.9% of patients.

Also, the newly discovered viruses known to cause gastroenteritis were detected, such
as astrovirus MLB2, primate bocaparvovirus1, Aichivirus A, cardiovirus, parechovirus A,
astrovirus VA4, cosavirus-F, and bufavirus-3. Our results showed 71% agreement (k =
0.445, P = 0.000) between multiplex Real-Time PCR, which is used as a routine diagnostic
test and metagenomics approach in the detection of viruses causing gastroenteritis in
clinical samples.
 2.

DYNAMICS OF FECAL MICROBIAL

COMMUNITIES IN CHILDREN WITH
DIARRHEA OF UNKNOWN ETIOLOGY AND
GENOMIC ANALYSIS OF ASSOCIATED
STREPTOCOCCUS LUTETIENSIS

DONG JIN, CHEN CHEN , LIANQING LI, SHAN LU, ZHENJUN

HTTPS://DOI.ORG/10.1186/1471-2180-13-141
 Although more than 200 viral, bacterial,
and parasitic causes of diarrhea have been identified to
date, only a few etiological agents cause the vast majority
of diarrheal diseases in children in the developing
world. These include rotavirus, diarrheagenic Escherichia
coli, Campylobacter jejuni, Shigella spp., non-typhoidal
Salmonella, Giardia lamblia, Cryptosporidium spp.
and Entamoeba histolytica. Unfortunately, a
large proportion of cases of diarrheal disease are of
unknown etiology. There are many reasons for this
problem, including fragility of causative agents, exacting
growth requirements, and lack of recognition of some
organisms as enteric pathogens.

Here, They used the previously described strategy of

16S rRNA gene polymerase chain reaction (PCR) and
sequencing technology to analyze quantitatively the
densities of different bacterial species in fecal samples
of patients with diarrhea of unknown etiology at different
times relative to hospital admission, and analyzed the
features of the dominant species.
 METHEDOLOGY
Children with diarrhea without antibiotic
treatment who were admitted to the
Children’s Hospital, Shanxi Province,
China from August 17 to 30, 2006 were
screened for enteric pathogens, including
Shigella, Salmonella, enterotoxigenic E.
coli, enteroinvasive E. coli (EIEC),
enteropathogenic E. coli (EPEC), Shiga-
toxin-producing E. coli,
enteroaggregative adherence E. coli
(EAEC), and common diarrhea viruses,
including group A rotavirus, human
calicivirus (HuCV), enteric adenovirus
(Adv) and human astrovirus (HAstV).
Total viral DNA and RNA were
extracted from fecal specimens
prepared in phosphate-buffered saline
at 10% (wt/vol) using the QIAamp
MinElute Virus Spin Kit (Qiagen, Hilden,
Germany) according to the
manufacturer’s recommendations.
HuCV, enteric Adv and HAstV were
detected by PCR . G. lamblia and Ent.
histolytica were detected using direct
microscopy with a saline preparation of
the specimen.
The clinical history and
physiological findings of each
patient were documented on
standardized case report forms.
Fecal samples from five healthy and
five hospitalized children at the
same location but with no apparent
diarrhea were analyzed as controls.
Libraries of the 16S rRNA gene
were constructed for each fecal
sample, with a minimum size of 100
analyzable sequences
ANALYZING DOMINANT FECAL BACTERIAL SPECIES BY
16S RRNA GENE SEQUENCE TECHNOLOGY
All fecal samples were collected in
triplicate; one for timely isolation and
detection of the enteric pathogens; one
stored at −20°C for 16S rRNA sequence
analysis; and one stored in 20% glycerol
at −80°C for isolation of the putative
The DNA was extracted from a
pathogens suggested by the 16S rRNA 200-mg fecal sample, which was
gene analysis. measured and adjusted to 100 ng/
μl of each sample for PCR. The
universal eubacterial primers 27
F519R (5’-agagtttgatcmtggctcag-3’
and 5’-gwattaccgcggckg ctg-3’)
were used to amplify a 500-bp
region of the 16S rRNA gene. LaTaq
polymerase (TaKaRa, Dalian, China)
was used for PCR under the
following conditions: 95°C for 5
min, followed by 20 cycles of: 95°C
for 30 s, 52°C for 30 s, and 72°C for 1
min; and a final elongation step at
72°C for 10 min.
The PCR products were extracted from sliced
gels and cloned into the pGEMR -T Easy
Vector System . The purified plasmid DNA
was used for sequence analysis. To verify the
repeatability, They then repeated the 16S
rRNA gene analysis of feces at admission for
nine children with diarrhea of unknown
etiology. The 16S rRNA gene sequences were
analyzed for chimeric constructs using the
Chimera Check program within the
Ribosomal Database Project.
Species-level identification was performed
using a 16S rRNA gene sequence similarity
of ≥99% compared with the prototype strain
sequence in the GenBank. Identification at
the genus level was defined as a 16S rRNA
gene sequence similarity of ≥97% with that
of the prototype strain sequence in the
GenBank, and the sequences were listed by
genus.
ISOLATION OF SUGGESTED FECAL-DOMINANT STREPTOCOCCUS

Strains of Streptococcus were

isolated from fecal samples using
KF Streptococcus Agar(Oxiod,
Hampshire, United Kingdom), and
identified using the MicroScan
Pulsed-field gel electrophoresis was
WalkAway SI 40 system(Dade
performed according to the protocol for
Behring,West Sacramento, CA,
Streptococcus suis . The DNA was
USA). The full length of the 16S rRNA
digested with 40 U SmaI. A dendrogram
gene sequence was obtained for
of isolates was drawn using
confirmation of identification
BioNumerics software (version 4.0,
Applied Maths BVBA, Belgium).
Clustering of patterns was performed
using the unweighted pair group with
arithmetic averaging (UPGMA).
GENOME SEQUENCING AND ANALYSIS OF STREPTOCOCCUS LUTETIENSIS

The genome of S. lutetiensis 033 isolated

from Patient 033 was sequenced using a
combination of 454 sequencings with a
Roche 454 FLX and paired end
Orthologous gene clusters were searched for
sequencing derived from the pUC18
using the orthoMCL pipeline. They clustered
library using an ABI 3730 Automated DNA
these orthologous genes according to their
Analyzer . presence or absence in different genome
The genome was predicted using sequences among Streptococcus spp., and
Glimmer software . then a phylogenic tree was constructed
using the neighbor-joining method.

Genome islands were defined as having

abnormal GC content with at least five
continuous genes. The homologous genes
within each island were compared with the
references using BLASTN with an e-value
cutoff at 1×10–5
 Nineteen of 33 children with diarrhea could not be etiologically
diagnosed by routine culture and polymerase chain reaction methods.
Eleven of 19 children with diarrhea of unknown etiology had
Streptococcus as the most dominant fecal bacterial genus at
admission.

Eight of nine children whom three consecutive fecal samples were

collected had Streptococcus as the dominant fecal bacterial genus,
including three in the Streptococcus bovis group and three
Streptococcus sp., which was reduced during and after recovery.

They isolated strains that were possibly from the S. bovis group from
feces sampled at admission, which were then identified as
Streptococcus lutetiensis from one child and Streptococcus
gallolyticus subsp.

RESULTS
pasteurianus from two children. They sequenced the genome of S.
lutetiensis and identified five antibiotic islands, two pathogenicity
islands, and five unique genomic islands. The identified virulence
genes included hemolytic toxin cylZ of Streptococcus agalactiae and
sortase associated with colonization of pathogenic streptococci.
RESULTS
 3

CLINICAL METAGENOMICS IS
INCREASINGLY ACCURATE AND
AFFORDABLE TO DETECT ENTERIC
BACTERIAL PATHOGENS IN STOOL
CHRISTY-LYNN PETERSON, DAVID ALEXANDER, JULIE CHIH-YU CHEN

https://doi.org/10.3390/microorganisms10020441
 Stool culture is the gold standard method to
diagnose enteric bacterial infections; however,
many clinical laboratories are transitioning to
syndromic multiplex PCR panels. PCR is rapid,
accurate, and affordable, yet does not yield
subtyping information critical for foodborne disease
surveillance. A metagenomics-based stool testing
approach could simultaneously provide diagnostic
and public health information.

Here, they evaluated shotgun metagenomics to

assess the detection of
common enteric bacterial pathogens in stool.
Methedology
 Results

Among analysis tools and

databases compared, KAT-SECT analysis provided the best sensitivity and specificity for all pathogens
tested compared to culture (91.2% and 96.2%, respectively). Where metagenomics detected a pathogen
in culture-negative specimens, standard PCR was positive 85% of the time. The cost of metagenomics
is approaching the current combined cost of PCR, reflex culture, and whole genome sequencing for
pathogen detection and subtyping. As cost, speed, and analytics for single-approach metagenomics
improve, it may be more routinely applied in clinical and public health laboratories.
Results
Thank You !