Variations in the Composition of Respiratory Microbiota

Variations in the Composition of Respiratory Microbiota Among Asthmatic and Non-asthmatic Subject

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.

Study Overview

• Status: Completed
• Conditions: Asthma
• Intervention / Treatment: Other: DNA extraction and 16S rRNA sequencing

Detailed 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).

• Study Type: Observational
• Enrollment (Actual): 54

Contacts and Locations

Study Locations

• Jordan
  • Amman, Jordan, 11931
    • Applied Science Private University

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 14 years and older (Child, Adult, Older Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

• Sampling Method: Probability Sample

Study Population

Asthmatic, non-smoker, aged 14 years-old living in Jordan

Description

Inclusion Criteria:

1. Asthmatic patients
2. healthy subjects
3. aged 14-year-old

Exclusion Criteria:

1. Smokers
2. patients reported having taken antibiotics for at least two months before study enrolment 3- patients who had other respiratory diseases or infections.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Asthmatic patients; Asthmatic patients aged 14-year-old, and more were included in the study. Knowing that patient with the age of 14-year is treated as adults at Al Bashir Hospital. Smokers, patients reported to have taken antibiotics for at least two months before study enrolment, and patients who had other respiratory diseases or infections were excluded from the study.	Other: 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.
Healthy subjects; Healthy subjects aged 14-year-old, and more were included in the study. Smokers, patients reported to have taken antibiotics for at least two months before study enrolment, and patients who had other respiratory diseases or infections were excluded from the study.	Other: 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.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
To identify the common microorganisms in the LRT among asthmatic patients and non-asthmatic subjects in Jordan	To asses the microbiome in the LRS between asthmatic patients and non-asthmatic by sequencing the 16s rRNA gene from the collected samples, after extracting the DNA	10 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
To identify a common microbial bioindicator that could be used as a diagnostic tool	To calculate a common microbial bioindicator that could be used as a diagnostic tool for an early diagnosis of asthma using 16s ribosomal RNA gene sequencing from sputum samples from the study population using statistical analysis and the biodiversity indicators	10 months

Collaborators and Investigators

• Sponsor: Applied Science Private University
• Investigators: Principal Investigator: Mohammad Al-Najjar, PhD, Faculty of Pharmacy, Applied Science Private University

Study record dates

Study Major Dates

• Study Start (Actual): December 1, 2019
• Primary Completion (Actual): May 1, 2020
• Study Completion (Actual): September 30, 2020

Study Registration Dates

• First Submitted: February 4, 2021
• First Submitted That Met QC Criteria: February 8, 2021
• First Posted (Actual): February 10, 2021

Study Record Updates

• Last Update Posted (Actual): February 10, 2021
• Last Update Submitted That Met QC Criteria: February 8, 2021
• Last Verified: February 1, 2021

More Information

Terms related to this study

• Keywords: Asthma; Haemophilus influenzae; Oral corticosteroids; Inhalers; Respiratory microbiota
• Additional Relevant MeSH Terms: Respiratory Tract Diseases; Immune System Diseases; Lung Diseases; Hypersensitivity, Immediate; Bronchial Diseases; Lung Diseases, Obstructive; Respiratory Hypersensitivity; Hypersensitivity; Asthma
• Other Study ID Numbers: 1800164

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No