Document Type : Original Research
Authors
1 Department of Pediatrics, Faculty of Medicine, Cairo University, Egypt.
2 Department of Clinical and Chemical Pathology, Faculty of Medicine, Cairo University, Egypt.
3 Department of Pediatrics, Al Anfoshy General Hospital, Alexandria, Egypt.
Abstract
Keywords
Introduction
Bronchial asthma is a chronic inflammatory airway disease characterized by hyper- responsiveness and episodic obstruction of the bronchi in response to various triggers. Asthma triggers include food, exercise and environmental factors such as polluted air and others (2). The determinants of the personal susceptibility to asthma are variable. The microorganism aggregates that dwell in the nasopharynx and the lung, as well as on the skin and mucosa of other systems (known as the microbiome) are vital for training of the immune system. They provide symbiotic support for regulation or symbiotic dysregulation of the immune response (3). The microbiome includes whole viruses, virus genomes, bacteria, archaea and other eukaryotes of the lungs and gut. They are linked in the gut-lung floral axis and both influence the T helper immune response (4). The microbiome-human interaction is defined by the types of food, the composition and concentration of micronutrients, substrates, the cellular metabolism, the resulting metabolic end-products as well as the geographic location (5). The microbiome dysbiosis (imbalance) and the metabolomics (how metabolites interact with the biological system) were noted to influence asthma severity and response to therapeutic agents (6). There has been no studies to date about the nasopharyngeal microbiome in Egyptian children. One study involved the nasal microbiome of adult males in rural and industrial areas (5), and several other research articles studied the gut microbiome in various conditions in adults (7–9). The work at hand aimed to study the composition of the nasopharyngeal microbiome among a cohort of Egyptian children with bronchial asthma, and to compare it to that of healthy controls of similar age and sex distribution.
Subjects and Methods
This cross sectional study was carried out at Cairo University Children’s Hospitals. The Pediatric Department Committee of Research and Higher Studies Research Committee of Faculty of Medicine, Cairo University approved the study, in compliance to Helsinki declaration guidelines (10).
Participants
The study included 60 children; 30 known asthmatics, aged 1-13 years old, diagnosed and following up in Allergy and Pulmonology Clinic at Cairo University Children's Hospital, as well as 30 age and sex matched non-asthmatic healthy children as a control group. Any child with acute upper respiratory tract infection at time of examination, or any other acute or chronic chest conditions apart from bronchial asthma were excluded. In addition, any child who received antibiotics in the two weeks preceding the swab was excluded. Asthmatic patients of any severity level were included (according to National Heart, Lung and Blood Institute 2017 guidelines) (11), but they were not in an exacerbation at the time of nasopharyngeal swabbing.
Methods
An informed consent was obtained from the parent/guardian of the included children. All included patients were subjected to detailed history taking, clinical and chest examination. Nasopharyngeal swabs were obtained from all children; patient and controls.
Nasopharyngeal swab procedure and culturing
The physician obtaining the swab wore sterile gloves and used a Dacron swab inserted slowly through the nostril to the posterior pharynx. Ideally, the swab was left in the posterior pharynx for 10 seconds before withdrawing. Optimal sampling and handling of the specimen was done to improve the rate of recovery of different microorganisms. Then the specimen was cultured on ordinary routine cultures for isolation and identification of the nasopharyngeal microbiome.
Microbiota identification
The nasopharyngeal swab was then cultured routinely, isolation and identification of the organisms was then done first by light microscopy and then by VITEK 2 system (bioMérieux, USA). The VITEK 2 system is a fully automated, extended-database system for bacterial and fungal identification and antibiotic sensitivity. Handling was minimized in a simple standardized inoculum. The inoculum was placed into the VITEK 2 Cassette, the VITEK 2 Card and sample were linked virtually. Once the Cassette was loaded, the incubation and reading of each card was managed by the system without any intervention. VITEK 2 could provide identification results in as little as 5 hours. The VITEK cards have 64 wells with various nutrients and biochemical tests. The inoculated microorganism reacts within the card creating a pattern of positive and negative reactions. This pattern is then compared automatically to a built-in library of database providing a name for the bacteria or fungus.
Statistical Analysis
For data analysis, the IBM SPSS V. 22.0 statistics program (IBM Corp., USA, 2013) was used. Data were tabulated as quantitative and qualitative data, where parametric and non-parametric data analysis was employed respectively. Standard student T-test was used for the comparison between the parametric data of the two groups. The calculated “t” was compared with the tabulated one at different levels of significance at the degree of freedom. Chi-square test of significance was used between the two groups as regards the qualitative data. A p-value less than 0.05 denoted statistical significance.
Results
|
Cases (N=30) |
Controls (N=30) |
P value |
Age (years) |
|
|
|
Mean +/- SD |
5. 67 ±3.3 |
5.1 ±3 |
0.45 |
Median (range) |
5 (1.1-13) |
4 (1.3-12) |
|
Gender (number, %) |
|||
Females |
13 (43.33%) |
12 (40%) |
0.5 |
Males |
17 (56.67%) |
18 (60%) |
|
Residence (number, %) |
|
|
|
Rural |
7(23.33%) |
11(36.67%) |
0.79 |
Urban |
23 (76.67%) |
19 (63.33%) |
Risk Factors |
Number |
% |
Positive family history |
16 |
53.33 |
Passive smoking |
15 |
50 |
Animal contact |
4 |
13.33 |
Perinatal History |
||
Delivery by Caesarian section |
22 |
73.33 |
NICU admission |
4 |
13.33 |
Exclusive breast feeding |
19 |
63.33 |
Asthma Severity |
||
Intermittent |
5 |
16.67 |
Mild persistent |
7 |
23.33 |
Moderate persistent |
10 |
33.33 |
Severe persistent |
8 |
26.67 |
Medications Used |
|
|
Short acting beta two agonists (SABA) as needed (prn) |
5 |
8.33 |
SABA (prn) & controller inhaled corticosteroids (ICS) |
16 |
26.67 |
SABA (prn) & controller ICS + monteleukast |
7 |
11.67 |
SABA (prn) & controller ICS + monteleukast + oral corticosteroids |
2 |
6.67 |
Hospitalizations for asthma |
17 |
56.67 |
*Asthma Severity, according to National Heart, Lung and Blood Institute, 2007 (11).
Table (3): The detected organism/combinations in the nasopharyngeal swab.
|
Cases (N =30) |
Controls (N =30) |
P value |
Overall P value |
CONS * |
0 |
5 (16.67%) |
0.045 |
0.007 |
Diphteroids |
3 (10%) |
4 (13.33%) |
0.68 |
|
Diphteroids, CONS* & Candida |
0 |
1 (3.33%) |
0.31 |
|
Diphteroids, K. rosea*, G. elegans* |
0 |
1 (3.33%) |
0.31 |
|
Diphteroids & CONS* |
0 |
1 (3.33%) |
0.31 |
|
Diphteroids & Anthracoids |
1 (3.33%) |
0 |
0.31 |
|
Diphteroids & Candida |
1 (3.33%) |
0 |
0.31 |
|
K. rosea* & Acinetobacter |
1 (3.33%) |
0 |
0.31 |
|
K. rosea* |
4 (13.33%) |
3 (10%) |
0.68 |
|
K. rosea* & Anthracoids |
0 |
1 (3.33%) |
0.31 |
|
K. rosea*, D. nishinomiyaensis*, K. sedentarius*, Diphteroids & G. elegans* |
1 (3.33%) |
0 |
0.31 |
|
K. rosea* & Diphteroids |
2 (6.67%) |
0 |
0.15 |
|
K. rosea* , Diphteroids & G. elegans* |
1 (3.33%) |
0 |
0.31 |
|
K. rosea* & , K. varians, D. nishinomiyaensis* & K. sedentarius* |
1 (3.33%) |
0 |
0.31 |
|
Staph. * aureus |
5 (16.67%) |
6 (20%) |
0.73 |
|
Staph. * aureus, CONS* |
1 (3.33%) |
1 (3.33%) |
NA |
|
Staph. * aureus & Diphteroids |
1 (3.33%) |
2 (6.67%) |
0.55 |
|
Staph. * aureus & Strept. * pneumoniae |
4 (13.33%) |
0 |
0.038 |
|
Strep. * pneumoniae |
3 (10%) |
4 (13.33%) |
0.68 |
|
Strep. * pneumoniae & Acinetobacter |
0 |
1 (3.33%) |
0.31 |
|
Strep. * pneumoniae & Strep. * pseudoporcinus |
1 (3.33%) |
0 |
0.31 |
|
Strep. * pneumoniae, CONS * |
0 |
1 (3.33%) |
0.31 |
CONS: Coagulase Negative Staphylococcus; D. nishinomiyaensis: Dermacoccus nishinomiyaensis; G. elegans: Granulitella elegans; K. rosea: Kocuria rosea; K. sedentarius: Kytococcus sedentarius; K. varians: Kocuria varians; N: number; Staph,: Staphylococcus; Strep.: Streptococcus.
Table 4: The frequency of individual organisms in the nasopharyngeal swab of the included children.
|
Cases (N =30) |
Controls (N =30) |
P value |
CONS (Coagulase Negative Staphylococcus) |
1 (3.33%)* |
9 (30%) |
0.009 |
Diphteroids |
9 (30%) |
6 (20%) |
0.27 |
Staphylococcus aureus |
11 (36.67%) |
8 (26.67%) |
0.28 |
Streptococcus pneumoniae |
8 (26.67%) |
6 (20%) |
0.38 |
Kocuria rosea |
10 (30%) |
4 (13.33%) |
0.06 |
Candida |
1 (3.33%) |
1 (3.33%) |
--- |
Granulitella elegans |
1 (3.33%) |
1 (3.33%) |
--- |
Anthracoids |
1 (3.33%) |
1 (3.33%) |
--- |
Acinetobacter |
1 (3.33%) |
0 |
0.31 |
Dermacoccus nishinomiyaensis |
1 (3.33%) |
0 |
0.31 |
*Data are presented as: number (percentage). Multiple organisms could be found within the same patient and thus the totals exceed 100%. N: number.
Table 5: Staph. aureus and Strep. pneumoniae distribution among the asthmatic children.
|
Staph. aureus and Strep. pneumoniae (N =4) |
None (N =26) |
P value
|
Age in years Mean ±SD Median (range) |
4.2 ±3.3 4 (1.1 - 13) |
5.9 ±3.2 5 (1.3 - 12) |
0.34 |
Gender Male Female |
2 (50%) |
15 (57.7%) 11 (42.3%) |
0.59 |
Animal Contact |
1 (25%) |
3 (11.5%) |
0.45 |
Smoking |
2 (50%) |
13 (50%) |
NA |
Family history |
1 (25%) |
3 (50%) |
0.35 |
Asthma Severity Intermittent asthma Mild persistent Moderate persistent Severe persistent |
3 (75%) 1 (25%) 0 0 |
17 (65.4%) 7 (26.9%) 2 (6.7%) 0 |
0.83 |
Medications SABA SABA & ICS SABA & ICS & Monteleukast SABA& ICS & Montleukast & oral corticosteroids |
1 (25%) 3 (75%) 0 |
8 (30.8%) 15 (57.7%) 1 (3.3%) 2 (7.7%) |
0.88 |
Hospitalization* for asthma |
2 (50%) |
15 (57.7%) |
0.59 |
Mode of delivery NVD CS |
2 (50%) 2 (50%) |
6 (23.1%) 20 (76.9%) |
0.24 |
NICU admission |
0 (0%) |
4 (15.4%) |
0.71 |
Feeding pattern Breast Bottle Breast & bottle |
2 (50%) 1 (25%) 1 (25%) |
17 (65.4%) 7 (26.9%) 2 (6.7%) |
0.55 |
CS: Caesarean section; ICS: inhaled corticosteroids; NVD: normal vaginal delivery; SABA: short acting beta 2 agonists; N: number; SD: standard deviation. Hospitalization*: history of hospital admission at least once.
Table 6: Distribution of Kocuria rosea among studied children with bronchial asthma.
|
Kocuria rosea (N =10) |
None (N =20) |
P value |
Age in years Mean ±SD Median (range) |
3.6 ±1.7 4 (1.1 - 13) |
6.7 ±3.9 6 (1.3 - 12) |
0.012 |
Gender Male Female |
6 (60%) 4 (40%) |
11 (55%) 9 (45%) |
0.55 |
Animal Contact |
2 (20%) |
2 (10%) |
0.41 |
Smoking |
5 (50%) |
10 (50%) |
0.65 |
Family history |
6 (60%) |
8 (40%) |
0.25 |
Asthma Severity Intermittent asthma Mild persistent Moderate persistent Severe persistent |
0 5 (50%) 1 (10%) |
5 (25%) 6 (30%) 5 (25%) |
0.02 |
Medications SABA SABA & ICS SABA & ICS & Monteleukast SABA& ICS & Montleukast & oral corticosteroids |
0 2 (20%) |
5 (25%) 5 (25%) 0 |
0.034 |
Hospitalization for asthma |
8 (80%) |
5 (25%) |
0.006 |
Mode of delivery NVD CS |
3 (30%) 7 (70%) |
5 (25%) 15 (75%) |
0.54 |
NICU admission |
4 (40%) |
0 (0%) |
0.01 |
Feeding pattern Breast Bottle Breast & bottle |
6 (60%) 3 (30%) 1 (10%) |
13 (65%) 5 (25%) |
0.95 |
CS: Caesarean section; ICS: inhaled corticosteroids; NVD: normal vaginal delivery; SABA: short acting beta 2 agonists; N: number; SD: standard deviation.
Table 7: Composition of microbiome nasopharyngeal swabs of asthmatic patients according to residence.
Organism (number in both urban and rural patients) |
Urban patients (N=18) |
Rural patients (N=12) |
P value |
||||
Number of urban residents having organism |
% of Urban residents with organism to total patients |
% of urban residents with organism to urban patients |
Number of rural residents having organism |
% of rural residents with organism to total patients |
% of rural residents with organism to rural patients |
||
Staph. aureus (11) |
6 |
54.5 |
33.3 |
5 |
45.5 |
41.7 |
0.643 |
Kocuria rosea (10) |
6 |
60 |
33.3 |
4 |
40 |
33.3 |
1.0 |
Diphteroids (10) |
7 |
70 |
38.9 |
3 |
30 |
25 |
0.429 |
Strept. pneumoniea (8) |
4 |
50 |
22.2 |
4 |
50 |
33.3 |
0.500 |
D. nishinomiyaensis (2) |
1 |
50 |
5.6 |
1 |
50 |
8.3 |
0.765 |
CONS staph (1) |
0 |
0 |
0 |
1 |
100 |
8.3 |
0.213 |
Kocuria varians (1) |
0 |
0 |
0 |
1 |
100 |
8.3 |
0.213 |
Kytococcus sedentarius (1) |
1 |
100 |
5.6 |
0 |
0 |
0 |
0.406 |
Acinetobacter (1) |
0 |
0 |
0 |
1 |
100 |
8.3 |
0.213 |
Granulicatella elegans (2) |
2 |
100 |
11.1 |
0 |
0 |
0 |
0.232 |
Candida (1) |
1 |
100 |
5.6 |
0 |
0 |
0 |
0.406 |
Anthracoids (1) |
0 |
0 |
0 |
1 |
100 |
8.3 |
0.213 |
Table 8: Composition of microbiome nasopharyngeal swabs of healthy controls according to residence.
Organism (number in both urban and rural patients) |
Urban Controls (N=17) |
Rural Controls (N=13) |
P value |
||||
Number of Urban residing controls having organism |
% of Urban residing controls with organism to total controls |
% of urban residing controls with organism to urban controls |
Number of rural residing controls having organism |
% of rural residing controls with organism to total controls |
% of rural residing controls with organism to rural controls |
||
Staph. aureus (11) |
6 |
66.7 |
35.3 |
3 |
33.3 |
23.1 |
0.469 |
Kocuria rosea (10) |
5 |
55.6 |
29.4 |
4 |
44.4 |
30.8 |
0.936 |
Diphteroids (10) |
4 |
50 |
23.5 |
4 |
50 |
30.8 |
0.657 |
Strept. pneumoniea (8) |
4 |
66.7 |
23.5 |
2 |
33.3 |
15.4 |
0.580 |
D. nishinomiyaensis (2) |
2 |
40 |
11.8 |
3 |
60 |
23.1 |
0.410 |
CONS staph (1) |
0 |
0 |
0 |
1 |
100 |
7.7 |
0.245 |
Kocuria varians (1) |
0 |
0 |
0 |
1 |
100 |
7.7 |
0.245 |
Kytococcus sedentarius (1) |
0 |
0 |
0 |
1 |
100 |
7.7 |
0.245 |
Acinetobacter (1) |
6 |
66.7 |
35.3 |
3 |
33.3 |
23.1 |
0.469 |
Granulicatella elegans (2) |
5 |
55.6 |
29.4 |
4 |
44.4 |
30.8 |
0.936 |
Candida (1) |
4 |
50 |
23.5 |
4 |
50 |
30.8 |
0.657 |
Anthracoids (1) |
4 |
66.7 |
23.5 |
2 |
33.3 |
15.4 |
0.580 |
Discussion
Conclusions
The composition of the nasopharyngeal microbiome in a group of asthmatic Egyptian children was significantly different from their healthy control peers. Coagulase negative staphylococci (CONS) were significantly higher in healthy controls, while a combination of Staph. aureus and Strep. pneumoniae was higher in asthmatic patients. Kocuria rosea was found to be more common among the younger asthmatics, and among children with a more severe asthma. Validation studies in various settings, and trials to assess if inducing eubiosis would favorably affect the disease course are recommended for future research.
Acknowledgment
The authors would like to acknowledge the heads, managers and workers in The Department of Clinical and Chemical Pathology, The Pediatric Allergy Unit and Pulmonology including Pulmonary Function Lab, as well as Children’s Specialty Hospital management (Faculty of Medicine, Cairo University) for making this work possible.
Author Contributions:
C.W.S.B.: Conceptualization, methodology, data analysis, writing original draft and editing. M.A.K.: Data analysis, writing original draft, supervising and revising. A.M.S.: Supervising laboratory work, data curation and analysis, investigation and methodology, visualization and revision of writing. M.A.: Data curation and analysis, investigation and methodology, revision of writing. M.G.: Sample collection, data curation and analysis, investigation and methodology. A.K.A.: Conceptualization, methodology, data analysis, revising and writing. All authors reviewed the final manuscript. All authors have read and agreed to the published version of the manuscript.
FUNDING
Authors declare there was no extramural funding provided for this study.
CONFLICT OF INTEREST
The authors declare no conflict of interest in connection with the reported study.