Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT04747184 |
| Other study ID # |
1800164 |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
December 1, 2019 |
| Est. completion date |
September 30, 2020 |
Study information
| Verified date |
February 2021 |
| Source |
Applied Science Private University |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
Induced sputum samples were obtained from 27 asthmatic patients and 27 non-asthmatic
subjects. Sequencing of the V4 region of 16S rRNA gene using Illumina MiSeq was performed,
followed by analysis of alpha and beta diversity.
Description:
The study was conducted over 10 months, from March to December 2019, with patients recruited
from Al Bashir public hospital,A convenient sample of 54 participants were approached (27
asthmatic patients and 27 healthy subjects). All the asthmatic patients were recruited from
an outpatient clinic at Al Bashir hospital. Study participants were informed about the nature
of the study and were included only after obtaining their consent verbally and voluntary.
Sociodemographic characteristics were collected from all study participants.
Specimens were collected according to the CDC guidelines for collecting respiratory diseases'
samples. All participants were asked to expectorate a sputum sample in special sterile tubes
indicated for sputum collection. Particular care was taken to avoid contamination of the
sputum sample with the saliva and post-nasal drip by asking the participants to rinse their
mouth with sterile water before inducing the sputum sample. Then the expectorated sputum
specimen was immediately homogenized and stored in the deep freezer (-80°C) for DNA
extraction for microbial gene sequencing and sequence analysis.
located in Amman, the capital city of Jordan. DNA extraction and 16S rRNA sequencing DNA
extraction using QIAamp® DNA mini kit (QIAGEN) was done under aseptic techniques in the
sterile room for all samples according to the manufacturer instructions. The DNA extracts for
the 54 samples collected (27 from asthmatic patients and 27 from healthy subjects) were
stored at -80° C in a sterile Eppendorf tubes until they were sent to the Molecular Research
(MR DNA) Lab (Molecular Research LP, Shallowater, TX, USA) for sequencing. Briefly, PCR
amplification of the 16s rRNA gene and its subsequent sequencing was done using Illumina. The
16s rRNA gene V4 variable region PCR primers ill27Fmod (AGRGTTTGATCMTGGCTCAG) /ill519Rmod
(GTNTTACNGCGGCKGCTG) with barcode on the forward primer were used in 30 cycles using the
HotStarTaq Plus Master Mix Kit (Qiagen, USA). After amplification, PCR products were checked
in 2% agarose gel to determine the success of amplification and the relative intensity of
bands.
The pooled and purified PCR product was then used to prepare the Illumina DNA library.
Sequencing was performed on a MiSeq following the manufacturer's guidelines. Sequence data
were processed using the MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). In summary,
the sequences were joined, depleted of barcodes then sequences <150bp removed, sequences with
ambiguous base calls were removed. Sequences were denoised, Operational taxonomic units
(OTUs) generated and chimeras removed. The OTUs were defined by clustering at 3% divergence
(97% similarity). Final OTUs were taxonomically classified using BLASTn against a curated
database derived from RDPII and NCBI (www.ncbi.nlm.nih.gov, http://rdp.cme.msu.edu). For
alpha diversity index, namely, Shannon index H, it was carried out in "Past Program" for data
analysis version 4.02, after filtering out the reading with relative abundance of <1%.
Shannon index varies from 0 for communities with only a single taxon to high values for
communities with many taxa (DeJong, 1975).
