study on the etiology of acute respiratory infection in children under 5 years old in nhatrang, 2009

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MINISTRY OF EDUCATION AND TRAINING

MINISTRY OF HEALTH

NATIONAL INTITUTE OF HYGIENE AND EPIDEMIOLOGY

VŨ VĂN THÀNH

RESEARCH ON THE CAUSES OF ACUTE RESPIRATORY
INFECTIONS AMONG CHILDREN UNDER 5 YEARS OLD IN
NHA TRANG CITY, 2009

Major :

Medical Microbiology

Code :

62.72.01.15

SUMMARY of
DOCTOR OF PHYLOSOPHY in MEDICINE
THESIS


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HA NOI – 2012
This doctoral thesis was completed at:

NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY

Supervisors:
1. Assoc.Prof., Dr. ĐẶNG ĐỨC ANH
2. Assoc.Prof., Dr. PHAN LÊ THANH HƯƠNG

Counter arguer 1: Assoc.Prof., Dr. Lê Thị Oanh- Hanoi Medical University
Counter arguer 2: Assoc.Prof., Dr. Lê Thị Luân- Center for Research and Production
of Vaccines and Biologicals
Counter arguer 3: Assoc.Prof., Dr. Nguyễn Vũ Trung- National Hospital for Tropical
Diseases

This doctoral thesis will be defended at the Examination Committee at Institute level
at:
NATIONAL INSTITUTE OF HYGIENE AND EPIDEMIOLOGY

On :

day month 6 year 2012

This doctoral thesis could be found at:
- The National Library.
- The National Institute of Hygiene and Epidemiology Library


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TABLE OF ABBREVIATIONS

cDNA

Complimentary deoxiribonucleotid acid

CFU

Colony Forming Unit

ELISA

Enzyme Linked Immunosorbent Assay

H. influenzae

Haemophilus influenzae

M. catarrhalis

Moraxella catarrhalis

MIC

Minimum Inhibitory Concentration

PBP

Penicillin Binding Protein

PCR

Polymerase Chain Reaction

RSV

Respiratory Syncytial Virus

RT-PCR

Reveser Transcriptation - Polymerase Chain Reaction

S. pneumoniae

Streptococcus pneumoniae

WHO

World Health Organization


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INTRODUCTION
Acute respiratory infections (ARI) are common infectious diseases among chidren in
developed and developing countries. According to the statistics of World Health
Organization (WHO), some 15 million children die each year globally, 95 percent of whom
are in developing countries and 4 million of them died because of ARI. Therefore, WHO
considers the identification and prevention of the origin of ARI as one of their foundational
stratergies in order to improve children’s health.
In Vietnam, the rate of ARI incidence and death are now high, which is ranked as the
first rate of the acute infection diseases. A wide variety of viruses, bacteria or parasites are
the origin of ARI. In developed countries, ARI is almost caused by viruses, which accounts

for 80-90% percent. The most common viruses are influenza, respiratory syncytial virus,
Rhinovirus và Adenovirus. In developing countries, the origin of ARI is bacteria, accounting
for 75 percent. The common bacteria are S. pneumoniae, H. influenzae và M. catarrhalis.
Since these bacteria are normal ones, and conditional pathogens, when they are being found,
it is uncertain to decide that they are the right pathogens. Hence, it is necessary to identify
the fundamental limit in order to decide whether the bacteria are nomal ones or pathogenic
ones. Although WHO has been attempted to prevent ARI in global scale, the rate of ARI
incidence and death decreases slowly because the antibiotic resistance of bacteria changes,
which restrains treating results.
Derived from those insights, we conduct this doctoral thesis on the topic:
“Research on the causes of acute respiratory infections among children under 5 years
old in Nha Trang City, 2009”
OBJECTIVES OF THE STUDY
1. Identifing the fundamental limits of some causes of ARI among children under 5
years old.
2. Identifing the causes of ARI among children under 5 years old and the antibiotic
sensitivity of some common bacteria.
3. Investigate several factors related to the causes of ARI among children under 5
years old.
RESEARCH SIGNICANCE
1. Indicate the fundamental limits of density of bacteria in nose and throat swabs, which is
basis for the analysis and evaluation of diagnostic tests of ARI in children.
2. Determine the distribution ratio of bacteria, viruses that may cause disease in healthy
children and children under the age of 5 who suffer from ARI.
3. Update information on the antibiotic sensitivity of some certain bacteria causing ARI in
children and point out some factors related to ARI in children.


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STRUCTURE OF THE STUDY
This disseartation consists of 125 pages, exclusive of references and appendix. There are 4
chapters, 38 tables, 10 graps, 3 pictures and 125 domestic and international reference
materials. The structure of the dissertation includes: 2 pages of Introduction, 35 pages of
Literature review, 26 pages of Methodology, 31 pages of Findings, 28 pages of Discussion,
2 pages of Conclusion, 1 page of Implications and 2 published articles related to the study.


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LITERATURE REVIEW
1.1 Anatomy features, physiology, immune respiratory system and normal virus system
in children.
1.1.1. Anatomy features:
Respiratory system is formed from day 20th – 22nd of the embryonic period. It is continued to
develop its structure and functions until the child is borned, and respiratory system starts
working. However, since its structure and fuctions are not fully developed, child’s
respiratory system continues to develop till adulthood.
1.1.2 Physiological features:
Due to the short air path, upper respiratory tract infection is easily spread to lower
respiratory tract. The airway resistance of respiratory system is very strong, but the
expansion of chest is weak. For respiratory tract disease, the mucosa has many blood
vessels, so congestion, edema, discharge happen ealisy, which makes the airway narrower.
At a result, air resistance increases, causing airway disorders.
1.1.3. Immune system protecting children’s repiratory system:
When pathogenic microorganisms enter our body through the respiratory tract, they
encounter the system of respiratory protection including mucosal barrier, humoral factors
and phagocytic cells.
1.1.4. Common bacteria system in children:
- Bacteria in the nose: in the nose there are some bacteria disguising themselves as

diphtheria and staphylococci. Among them, aureus is noticable. Up to 20-50% healthy
people carry aureus in theỉ nose, and this proportion is higher among people working in the
hospital environment.
- Bacteria in the nose and throat: in the throat, there is a variety of number and species of
bacteria which are spread from the mouth, such as S.pneumoniae, H. influenzae, M.
catarhalis, S. viridans. However, depending on their location, pathogenic bacteria
commonly cause sore throat and rheumatic heart disease. Particularly, almond gland usually
has Streptococcus group A.
- Bacteria in the trachea and bronchi: due to the structural characteristics, physiology has
mucous substances and macrophages, so there is no bacterium in the lower respiratory tract.
1.2. The causes of ARI among children under 5 years old and the antibiotic sensitivity
of some common bacteria
1.2.1.1. The real situation of researching on ARI among children under 5 years old
1.2.1.1. Research on ARI among children under 5 years old in the world


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ARI among children under 5 years old is ranked at the hightest rate of incidence and death
worldwide. The mortality rate of ARI in developing countries is 30-5- higher than that in
developed countries. Study shows that: in developed countries, the origin of ARI is almost
viruses, which accounts for 80-90 percent. In developing contries, the origin of ARI is
bacteria accounting for 75%.
1.2.1.2. Research on ARI among children under 5 years old in Vietnam:
In Vietnam, the rate of ARI incidence and death is mainly among children under 5 the age
of five. According to a report of a national programme about preventing ARI in 2000, the rate of
ARI among children is 65,8%, which is the highest rate among the common disease in the
community.
1.2.2. The original virus causing ARI among children under 5 years old
1.2.2.1. Influenza virus:

Pathogenic role: influenza viruses are transmitted from person to person via the respiratory
tract. Some common clinical symptoms are light fever, sneezing, headache, cough, eccrine a
lot. In young children, some symptoms are high fever, convulsions, inflammation of the
stomach - intestines. In infant, symptoms are much more serious, such as myocarditis,
pneumonia, which can lead to encephalitis and even death.
Diagnostic from laboratories:
- How to collect samples: specimens should be taken in the onset of disease. The best
specimens for influenza diagnosis are nasal throat swabs combined with mouth and throat
swabs.
- The specimen for serological diagnosis: blood in the acute phase is taken as soon as
possible. After 14-28 days, blood in the recovery phase is taken. Serum is stored at -20 0C
until it is tested.
- Methods of diagnosis:
+ Isolation and identification of kinds of viruses: swabs in environment are transplanted into
the amniotic cavity of the embryos of 9-day-old chickens. They are incubated for 48 hours
at 330C, and at 40C overnight. Amniotic fluid is collected, HA titers is determined, and if
results is positive, HI is conducted to identify the type and subtypes.
+ Discover genetic material of viruses: the genetic material of viruses is the negativestranded ARN virus. Molecular techniques used to discover genetic material of influenza
viruses are RT-PCR.
+ The immunological techniques used in plasma diagnostics and influenza research:
complement fixation reaction, erythrocyte inhibitory reaction, ELISA techniques.
1.2.2.2. Respiratory syncytial virus:


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Pathogenic role: RSV is spread through contact with droplet secretions containing viruses.
Children from 6 weeks to 6 months old are usually infected by this disease. The symptoms
in onset phase are some symptoms of upper respiratory tract infection, such as fever, cough,
and runny nose. For older RSV infected children, the symptoms are usually like having a

cold, or there are very few symptoms. Therefore, the difficulty in clinical diagnosis occurs.
Diagnostic from laboratories: swabs are nose and throat liquid which is translated in the cell
recptors in order to find giant cell. In addition, diagnostic methods are also determined by
immunofluorescence, PCR.
1.2.2.3. Adenovirus:
Pathogenic role: viruses that cause acute infection have some characters such as: a short
incubation period, prolonged excretion virus, mild disease. However, pneumonia in young
children may cause death. The viruses do not multiply themselves, but they long- live exist
in cells. When our body's resistance is reduced, the viruses multiply themselves and cause
disease.
Diagnostic from laboratories: use Hep 2 cells which is cell-specific receptor for Adenovirus.
The plastic blade used to transplant Hep 2 cells are infected by the swabs. If cells are
damaged more than 25%, PCR or immunofluorescence is conducted to identify the
Adenovirus.
1.2.2.4. Rhinovirus:
1.2.2.4. Rhinovirus:
Role pathogens: rhinovirus enters the body through inhaling the particles of respiratory
secretions of an infected person or indirectly through hands, hand towels, furniture, toys of
infected children’s respiratory secretions containing viruses. The viruses enter the body
through the upper respiratory tract and are often limited here.
Diagnostic from laboratories: the laboratory diagnosis relies on virus isolation from nasal
and throat secretions, in culturing cells. Use PCR techniques to identify specific gene of the
virus.
1.2.3. The original bateria causing ARI among children under 5 years old
1.2.3.1. S. pneumoniae: Streptococcus pneumoniae normally resides in the human
nasopharynx in a nondisease state, with high rate of 40 to 70%. Bacteria can cause
respiratory infection, typically they can cause pneumonia. Pneumonia caused by S.
pneumoniae occurs after respiratory is infected by viruses or chemicals. S. pneumoniae also
causes ear infections, sinusitis, sore throats, sepsis and meningitis in children.
Laboratory diagnosis:

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* Gram stain: nose and throat swabs are used to identify the causes. Because many types of
bacteria are mixed toghether, if bacteria are identified, the bacteria may belong to the
normal bacteria generation.
* Cultural isolation: base on the rules of the World Health Organization.
Criteria for identification of pneumococcus:
- Bacteria on 5% blood agar environment: small, wet, concave in the middle, alpha
hemolytic.
- The nature of bacteria: diplococcus in the shape of a candle or a pair of glasses which
catch color Gram (+) and is sensitivity to optochin (diameter of sterile cirlce ≥ 14 mm) or is
less sensitive to optochin, but can be soluble in bile salts.
Not pneumococcal when:
- They are not sensitive to optochin
- The inhibition is developed by optochin with the diameter of <14mm, and they cannot be
soluble in bile salts.
1.2.3.2. H. influenzae:
Pathogenic role: H. influenzae is obligatory parasites in the respiratory mucosa of human.
About 75% of healthy children carry H. influenzae in their nose and throat as members a
normal bacteria generation. This rate is lower in adult. H. influenzae is classified into two
main groups: typeable (encapsulated) and nontypeable (nonencapsulated). Nontypeable
often causes mild infection in the upper respiratory tract such as tonsillitis, otitis media.
Typeable causes serious infection such as meningitis, blood infection.
Diagnostic from laboratories:
* Gram stain: estimate the number of infected cells and bacteria inside and outside the cells
by +, + +, + + +.
* Culturing and isolation: base on the rules of the World Health Organization.
Criteria for identifying H. influenzae:

- Small coccobacille, catch color Gram (-), bacteria in light gray, iridescent, cannot cause
hemolytic.
- Homogeneous development on selected environment.
- X and V factor are needed for development.
- The type of biological biochemical reactions: ODC, URE and IND.
- The serotype: most pathogenic strains are type b.
1.2.3.3. Moraxella catarrhalis:
Pathogenic role: M. catarrhalis is one of three common origins which cause ear infection in
children, accounting for 10-15%. M. catarrhalis are bacteria that cause opportunistic
infections and reside in the upper respiratory tract of healthy children. Middle ear infection
often occurs when there is an increase of direct contact with bacteria from the resident


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neighborhood; Pneumonia starts from small bronchial branches of the lung. 85% of M.
catarrhalis strains can be isolated in the United States, gave birth to β - lactamase.
Diagnostic from laboratories:
* Gram stain: swabs are fluid, pus, blood, depending on each type of infection. Gram stain has
an important meaning in the diagnosis of M. catarrhalis.
* Culture and isolation: base on the rules of the World Health Organization.
Criteria for identifying M. catarrhalis are:
- Diplococcus catch Gram color (-)
- Do not grow on Mac Conkey
- There is no resolution of sugars
- ONPG (-), oxidase (+), catalase (+), DNase (+)
1.2.4. The real situation of drug-resistance of bacteria causing ARI in children under 5 years
old and techniques to identify the rate of antibiotic sensitivity
1.2.4.1. The real situation of drug-resistance of bacteria causing ARI in children under 5
years old

In Vietnam, many studies on the rate of antibiotic sensitivity of bacteria causing ARI,
especially, antibiotics being recommended to use in ARI treatment showed that antibiotic
resistance rates have increased show that the rate of antibiotic resistance is increasing.
1.2.4.2. Antibiotic techniques:
• disk diffusion antibiotic sensitivity testing (Kirby-Bauer):
* Principle: different strains of bacteria have different rate of sensitive to antibiotics at
different levels and are manifested in the difference in diameters around the sterile circular
of antibiotic paper, which contain determined attenuation when the contact between the
bacteria with antibiotics occurs.
* Purpose: Study on the antibiotic sensitivitive of bacteria helps clinical direction have right
treatment with specific antibiotics, and help contribute to evaluation of the situation of drugresistance of bacteria.
* Steps to follow: base on the rules of World Health Organization
♦ Techniques to identify Minimum Inhibitory Concentration: MI
* Principle: concentration increases in the culture medium. When it reaches a certain
concentration, it will inhibit the growth of bacteria and it can be determined by the human
eyes.


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* Purpose: techniques to determine accurately the MIC of the growth of bacteria in a culture
medium. Thereby, the antibiotic sensitivity of bacteria is determinined.
* Steps to follow: base on the rules of the World Health Organization.
Chapter 2. SUBJECTS AND METHODOLOGY
2.1. Subjects of the study
- Proved group includes 350 clinical specimens of nasopharyngeal juice taken from 350
under 5-year-old healthy children in Vinh Thanh and Vinh Phuong Commune, Nha Trang
City.
- Diseased group includes 441 clinical specimens of nasopharyngeal juice taken from 441
under 5-year-old children sufferring RID in Pediatrics of Khanh Hoa Hospital.

2.2. Methods
2.2.1. Research designing: Descriptive and cross-sectional methods with an analysis were
used in the survey.
2.2.2. Content of the study
2.2.2.1. For 350 clinical specimens of nasopharyngeal juice taken from under 5-year-old
healthy children
PCR method and quanlitative PCR to identify bacteria available on the clinical specimens of
nasopharyngeal were used.
2.2.2.2. For 441 clinical specimens of nasopharyngeal juice taken from under 5-year-old
children sufferring RID
- Culture, isolation and original cause determination techniques were used.
- PCR method and quanlitative PCR for the original cause identification were included.
- Levels of sensibility to antibiotic were identified thanks to Minimum Inhibitory
Concentration (MIC) method.
- PCR was used to identify S. pneumoniae and H. Influenzae which contained protesting
genes.
- Multiplex RT-PCR and Heminested PCR were applied for causal virus’s identification.
2.2.3. Techniques used in the study
2.2.3.1. Techniques for taking medical samples: following the routine regulations of WHO.
2.2.3.2. Culture, isolation and original cause determination techniques: following the
routine regulations of WHO and Institute of Tropical Medicine, Nagasaki University, Japan


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2.2.3.3. S. pneumoniae, H. influenzae, M. catarrhalis identification by PCR and quanlitative
PCR methods.
* Objectives of the methods: These methods are to establish a rapid and sensitive
experiment and high specificity for S. pneumoniae, H. influenzae and M. Catarrhalis in The
clinical specimens of nasopharyngeal juice taken from healthy children and under 5-yearold children sufferring RID based on PCR system; This experiment will be applied in the

laboratories of diagnosis of bacterial respiratory diseases in Vietnam. Multi-primer is to
detect such three types of bacteria as S. pneumoniae, H. influenzae, M. catarrhalis and three
quanlitative PCR reations based on the principle of Syber Green that is used to quantify
each type of bacteria in the samples.
* The pathogenous bacteria: The pathogenous bacteria that usually present in respiratory
samples are collected and used as reference to the specificity analysis.
* The clinical specimens of nasopharyngeal juice
There are 350 clinical specimens of nasopharyngeal juice taken from 350 under 5year-old healthy children in Vinh Thanh and Vinh Phuong Commune, Nha Trang City and
441 clinical specimens of nasopharyngeal juice taken from 441 under 5-year-old children
sufferring RID in Pediatrics of Khanh Hoa Hospital. Samples were taken according to the
routine regulations of WHO. They are immediately distilled in 1ml of STTG environment
(skimm milk, tryptone soy, glycerol and glucose) and preserved at minor 800C until they are
tested.
* DNA seperation: it is followed the commercial kit process QIA mini kit. DNA of
reference of pathogenous bacteria was also isolated by QIAamp kit. After seperation, the
DNA is determined by the concentration and purity of its optical gauges Eppendorf AG of
Germany. The DNA samples are preserved at minor 300C.
* Primers design:
Three sets of primers are used for primers PCR and quanlitative PCR; of which there
are two sets of primers to detect S. pneumoniae and M. catarrhalis that have been reported
in previous studies and one set of primers to detect H. influenzae designed by laboratory
staff. All of them are linked and compared the similarity thanks to MEGA 4.0 software and
new sequences that were chosen to be at least 1-4 first of bases in the three minutes of
'specific for H. influenzae. Then the primer sets were tested again by BLAST to determine
specificity.
•

Implement the PCR and quantitative PCR to determine S. pneumonia, H. influenza, M.
catarrhalis:

Firstly, implement the PCR reaction with multi baits to detect concurrently S.
pneumonia, H. influenza, M. catarrhalis. The clinical specimens which are negative for
multi-bait PCR are reevaluated by single-bait PCR. The positive clinical specimens are


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tested with quantitative PCR to determine the number of three kinds of bacteria that are S.
pneumoniae, H. influenzae, M. catarrhalis.

•

Identify the result: As for the group of clinical specimens of which the bacteria are
considered positive; the bacteria of group of clinical specimens which are more than 10 7
CFU per 1 ml are considered positive.

2.2.3.4 Multiplex RT-PCR and Heminested PCR method to identify the root of virus
- The bait system is used to detect the hereditary material of the virus causing respiratory
diseases through Multiplex RT-PCR and Heminested PCR method
- Set of biological products to separate ARN: QIAamp viral RNA Mini Kit, 250 preps, Cat
No 52904, Qiagen.
- Set of biological products used for RT-PCR method: QIAGEN Onestep RT- PCR kit, Cat.
No 210212.
- RNase inhibitor (40U/µ) – Invitrogen.
♦ Multiplex RT-PCR method
The clinical specimens of nasopharyngeal juice are collected at the time when
children are taken to hospital and immediately put in the tube of transporting environment.
The clinical specimen samples are preserved at minor 800C until they are tested. The
separation of AND in the clinical specimen samples is done by the kit of QIAamp Viral
ARN minikit (QIAGEN Inc., Valencia, CA)

Use four multiplex PCR reactions to detect 13 viruses causing respiratory diseases in
the clinical specimens: Rhinovirus, Respiratory Syncytial Virus, flu virus A and B, flu virus
tube 1-4, Humanmetapneumovirus, Adenovirus, Bocavirus, Coronavirus 229E & OC 43.
•

Separate AND: Following the commercial kit process (Republic of Germany)

•

Multiplex RT-PCR method:

•

Tait: The use of bait depends on PCR reactions

•

•

PCR reaction: 940C per 5 minutes, 940C per 30 seconds, 560C per 30 seconds, 720C per
90 seconds X 35 cycles , 720C per 5 minutes; be kept at 40C.
Electrophoresis of PCR products:

Implement the electrophoresis of PCR products on the 2% agar with ethidium
bromide ( 10...1/100ml of agarose fluid), voltage of 100V in 30 minutes. Use the molecular
weight scale 100bp. The AND bands can be observed and shot under the UV ray.


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• Identify the rerult:
Rhinovirus – 254 bp
Adenovirus – 193 bp
• The result is only read when:
The positive control is flu virus, Rhinovirus, Adenovirus, RSV...
The negative control reaction, the negative control separation: negative
Negative: There appear the PCR products in the specific position and there is no
presence of products.
Positive: Specific PCR products have the same length as the positive control.
♦ Heminested PCR method:
• Primers:: The use of bait depends on PCR reactions.
• PCR reaction: 940C per 5 minutes, 940C per 30 seconds, 560C per 30 seconds, 720C per
90 seconds X 35 cycles , 720C per 5 minutes; be kept at 40C.
• Identify the result:
- A-flu virus – 151 bp
- Respiratory Syncytial Virus – 201 bp
- The samples which are positive for 2 times of PCR are considered positive
2.3.5 Methods to determine the Minimum Inhibitory Concentration of antibiotics towards
the bacteria (MIC):
- Puposes: This technique aims at determining exactly the minimum concentration of
antibiotics which can inhibit the development of one race of bacteria in culturing
environment.
- Principles: The concentration of antibiotics increase in the culturing environment, at a
certain level, it will inhibit the development of bacteria and this can be determined by eyes
- Race of bacteria needs determining MIC:
152 races of S. pneumonia and 104 races of H. influenza have been identified and
made pure. Choose randomly 89 races of S. pneumonia and 37 races of H. influenza to test
MIC and PCR method to determine the gene of drug resistance.
- Steps: Following the regulations of World Health Organization

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- Reading the result: Read the result from the agar plate with the lowest antibiotic
concentration. The concentration of MIC is determined in the environmental plate in which
the bacteria are inhibited to develop. MIC result is compared with antibiotic boundary
concentration in order to divided into three levels: sensitive, anti or mediate.
2.4. Data collection and data analysis
The results will be collected and analyzed thanks to the methods of medical statistics by
using SPSS 16.0 software, Microsoft Office Excel 2009. The statistical algorithm was used
in converting into percentage. Chi-square test, Fisher′s exact test was used to test the
relationship between quantitative variables and statistical significance level at 95%
(p<0.05). The results will be illustrated by means of charts and tables.
2.5. Ethical issues in research
The research was approved by the Ethics Council of Hygiene and Epidemiology Institute. It
is carried out in accordance with the medical principles and ethics. The relatives of research
subject were clearly explained the research objectives and had agreement in participation.
The private information was absolutely confidential. The fee of experiments for the clinical
specimens of nasopharyngeal juice is freely covered all. The research subjects are looked
after and treated as other patients. The experiment results are used for diagnosis, prevention
and treatment.
Chapter 3. RESEARCH FINDINGS
3.1. Findings from determining the background restrictions of some causal bacteria
causing RID in under 5-year-old children.
Table 3.1. Findings from determining the background restriction of S. pneumoniae:

Groups of children

Numbers
(n)

Rates (%) having S. pneumoniae
≥ 106

≥ 107

105
CFU/1ml

CFU/1ml

CFU/1ml

≥

Pneumonia -X rays (+)

105

92,38

72,38

50,48

Bronchitis

84

73,81

57,14

20,24

Healthy

176

75,57

48,3

10,8

=0,1

=0,0001

=0,0001

P

At concentration ≥ 105 CFU/1ml of the medical waste, the rates of S. pneumonia
subdivided from three groups of children were different, which did not have statistical
meanings with p > 0.05. At concentration ≥ 107 CFU/1ml of the medical waste, the
differences of the rates of S. pneumoniae subdivided from 3 groups of children had the
statistical meanings with p < 0.05. Among them, the inadequacy of the rates of S.

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pneumoniae subdivided from the pneumonia-X rays (+) group and the healthy group was
4.8 times, bronchitis and healthy groups was 1.2 times.

Table 3.2. Findings from determining the background restriction of H. influenzae:

Groups of children

Numbers
(n)

Rates (%) having influenzae
≥ 106

≥ 107

105
CFU/1ml

CFU/1ml

CFU/1ml

≥

Pneumonia -X rays (+)

139

89,93

67,63

28,78

Bronchitis

112

93,75

68,75

41,96

Healthy

110

90,0

60,91

20,0

=0,503

=0,405

=0,002

P

At concentrations ≥ 105 CFU/1ml and ≥ 106 CFU/1ml of the medical waste, the rates
of H. influenzae subdivided among three groups of children are different, which did not
have statistical meanings with p > 0.05. At concentration ≥ 107 CFU/1ml of the medical
waste, the differences of the rates of H. influenzae subdivided from 3 groups of children had
the statistical meanings with p < 0.05. Among them, the inadequacy of the rates of S. H.
influenzae subdivided from the bronchitis and healthy groups was 2.1 times.
3.2. Findings from determining the cause of RID in under 5-year-old children.
3.2.1. Findings from determining the causal bacteria causing RID in under 5-year-old
children
3.2.1.1. Findings from determining bacteria in under 5-year-old healthy children:
Table 3.4. Findings from determining bacteria in under 5-year-old healthy children.
Kinds of bacteria
S. pneumoniae
H. influenzae
M. catarrhalis

Negative
Numbers
Rates
(n)
(%)
174
49,7
240
68,6
147

42,0

Positive
Numbers Rates (%)
(n)
176
50,3
110
31,4
203
58,0

Total
350 (100,0)
350 (100,0)
350 (100,0)


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Of total 350 medical waste samples in sore-throats of under 5-year-old healthy
children, it was determined the rates of three kinds of bacteria: S. pneumonia: 50.3%, H.
influenza: 31.4% and M. catarrhalis: 58.0%.

3.2.1.2. Findings from determining the causal bacteria causing RID in under 5-year-old
children:
Table 3.9. Rates of causal bacteria causing RID in 441 medical waste samples of under 5year-old children:
Medical wastes

Numbers

Rates (%)

Bacteria included medical wastes

328

74,4

S. pneumoniae

171

38,8

H. influenzae

227

51,5

M. catarrhalis

139

31,5

The rate of medical wastes determining the causal bacteria of the disease was 74.4%,
the rate of medical wastes excluding the bacteria was 25.6 %. In which, H. influenzae
accounted for 51.5%, S. pneumonia: 38.8% and M. catarrhalis: 31.5%.

Table 3.10. Rates of H.influenzae type B subdivision in under 5-year-old children
catching the RID:
Order

H. influenzae

Numbers (n)

Rates (%)

1

No-cover

100

96,1

2

Cover type b

4

3,9

3

Total

104

100

Of total 104 types of H. influenza causing RID, mainly, these were no-cover type,
accounting for 96.1 %, while there were only 4 cover type b H.influenzae, making up 3.9%.
Table 3.11. Rates of autosome process in two types of bacteria in children catching RID
according to ages:
Bacteria
Ages

SP + HI
Negative

Positive

SP + MC
Negative

Positive

MC + HI
Negative

Positive


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< 12 months

59
73
65
67
64
68
(41,5%)
(24,4%)
(33,2%)
(27,3%)
(38,8%)
(24,6%)
12 – 23
41
151
77
115
56
136
months
(28,9%)
(50,5%)
(39,3%)
(46,9%)
(33,9%)
(49,3%)
24 – <60
42
75

54
45
72
63(25,8%)
months
(29,6%)
(25,1%)
(27,5%)
(27,3%)
(26,1%)
Total
142
299
196
245
165
276
(100,0)
(100,0)
(100,0)
(100,0)
(100,0)
(100,0)
P
=0,0001
=0,242
=0,002
The rates of autosome process in two types of bacteria causing RID was mainly in
the age of 12-23 months, in which the autosome process of S. pneumoniae and H.
influenzae accounted for 50.5%; M. catarrhalis and H. influenzae made up 49.3%. The

differences among ages had statistical meaning with P < 0.05; the autosome process of S.
pneumoniae and M. catarrhalis accounts for 46.9%, these differences among ages had no
statistical meanings with p > 0.05.
3.2.1.3. A comparison of findings from determining causal bacteria in healthy children to
that of RI infected children:

Figure 3.4. A comparison of S. pneumoniae rate in healthy children to that of RI infected children according to ages

The rate of determined S. pneumoniae in healthy children was mainly at the age of 2460 months, accounting for 48.9 %, while in the infected group, it was mainly at the age of
12-23 months, making up 52.6 %. The differences of the rates of determined S. pneumoniae
according to ages between the healthy and infected children had statiscal meanings with P <
0.05.


16

3.2.1.4. Findings from levels of sensibility to antibiotic of bacteria types causing RID in
under 5-year-old children:
Table 3.17. Levels of sensibility to antibiotic of 89 types of S. pneumoniae causing RID
in under 5-year-old children:
Order

Antibiotic

Ratess (%)

Vacillation
scales

MIC

50

MIC
90

S

I

R

1

Penecillin G

0,032-4

0,5

2

7,9

83,1

9,0

2

Ampicillin

0,032-16

1

4

16,9

77,5

5,6

3

Amoxycillin/Cla

0,008-8

0,5

2

85,4

10,1

4,5

4

Cefuroxime

0,032-64

4

16

36,0

5,6

58,4

5

Cfotaxime

0,016-4

0,5

2

61,8

19,1

19,1

6

Erythromycin

0,004->128

>128

>128

10,1

5,6

84,3

7

Chloramphenicol

0,5-32

4

8

65,2

23,6

11,2

8

Co - trimoxazole

4->128

64

>128

5,6

0

94,4

9

Clindamycin

0,004->128

128

>128

20,2

0

79,8

10

Ofloxacin

0,5-4

2

2

92,1

7,9

0

11

Tetracycline

0,125-128

32

64

11,2

0

88,8

12

Rifampicin

0,032-0,25

0,125

0,25

100

0

0

Of 152 types of S. pneumoniae determined in under 5-year-old children catching
RID, I randomly chose 89 types to experiment with antibiotic, using MIC method. The
findings pointed out that the rates of S. pneumoniae being sensitive to rifampicin antibiotic
was 100%, to ofloxacin antibiotic was 92.1%; however, others resisted co-trimoxazole
94.4%, tetracycline 88.8%, erythromycin 84.3%, penicillin G 9.0%, but in intermediary
state, it was 83,1%. Moreover, it resisted ampicillin 5.6% but in the intermediary state it was
77.5%.

17

Table 3.18. Rates of S. pneumoniae type which cause RID have sudden mutation pbp
gene resisting penicillin:
STT

Rate of pbp sudden mutation

Numbers (n)

Rates (%)

1

3 pbp sudden mutation

139

91,5

2

1 pbp sudden mutation

11

7,2

3

2 pbp sudden mutation

1

0,7

4

3 pbp normal

1

0,7

5

Total

152

100

Of 152 S. pneumonia types causing RID in under 5-year-old children were
subdivided, I used PCR technique to determine the sudden mutation of pbp gene protesting
penicillin. The findings figured out that 139 types had 3 pbp, accounting for 91.5%, 11
types had 1 pbp, making up 7.2%, 1 type had 2 pbp, namely 0.7%, 1 typ had 3 pbp normal,
accounting for 0.7%.
Table 3.19. Rate of S. pneumoniae having antibiotic resisting macrolide genes:

Order

Antibitic resisting macrolide
genes

Numbers (n)

Rates (%)

1

Negative

5

3,3

2

1 gene ermB

93

61,2

3

1 gene mefA

5

3,3

4

2 gene ermB+mefA

49

32,2

5

Total

152

100,0

Of
152 types of S. pneumoniae causing RID that were subdivided, there are 5 types that did not
have the antibiotic resisting macrolide gene, accounting for 3.3%, 93 types had 1 ermB
gene, accounting for 61.2%, 5 types had 1 mefA gene, making up 3.3% and 49 types had
both ermB and mefA genes , making up 32.2%.


18

Table 3.20. Levels of sensibility to antibiotic of 37 types of H. influenzae causing RIDin
under 5-year-old children:

Order

Antibiotic

MIC
vacillating
scales

MIC
50

MIC
90

Rates (%)
S

I

R

1

Ampicillin

0.125 - 64

>64

>64

40,5

0

59,5

2

Amoxycillin/Cla

0.125 - 8

1

3

97,3

0

2,7

3

Ampicillin/Sul

0.063 - 16

0,5

8

78,4

0

21,6

4

Cefuroxime

0.5 - 64

2

32

63,8

16,7

19,4

5

Cfotaxime

0.008 - 2

0,032

0,5

100

0

0

6

Ceftriaxone

0.004 - 0.25

0,008

0,125

100

0

0

7

Ceftazidime

0.063 - 2

0,125

1

100

0

0

8

Cefepime

0.063 - 2

0,125

1

100

0

0

9

Azithromycin

1,0-4,0

2

2

100

0

0

10

Clazithromycin

2,0-16

4

8

91,2

8,8

0

11

Chloramphenicol

0,25-16

1

16

56,8

0

43,2

12

Tetracycline

0,25-32

0,5

16

62

0

38

13

Ciprofloxacin

0,008-0,016

0,016

0,016

100

0

0

14

Co - trimoxazole

0,5->128

>128

>128

13,5

0

86,5

15

Rifampicin

0,25-1

0,5

0,5

100

0

0

H.influenzae was totally sensitive to Cfotaxime, ceftriazone, ceftazidime, cefepime,
rifampicin antibiotics; resisted co – trimoxazole 86.5%, ampicillin 59.5%, chloramphenicol
43.2% antibiotics.

Table 3.21. Rates of H. influenza type having protesting β – lactam genes:


19

STT

β- lactam resisting genes

Numbers (n)

Rates (%)

1

Negative

53

51

2

TEM – 1

51

49

3

ROB – 1

0

0

4

Total

104

100

The findings from showed that there were 53 types that did have β- lactam resisting genes,
accounting for 51%, 51 types had blaTEM-1 gene resisting β- lactam antibiotic, accounting
for 49%, no type had blaROB-1 gene .
3.2.2. Findings from determining the causal viruses of RID in under 5-year-old children:
Table 3.23. Rate of causal viruses causing RIDin under 5-year-old children:
Medica wastes

Numbers

Rates (%)

Viruses-included
medical wastes

307

69,6

Influenza virus type A

69

15,6

RSV

96

21,8

Rhinovirus

124

28,1

Asian influenza virus

28

6,3

Adenovirus

25

5,7

HMPV

18

4,1

Bocavirus

12

2,7

Influenza virus type B

2

0,5

307 medical wastes were determined to include viruses, of which influenza virus type
A accounts for 15.6 %, RSV: 21.8 %, Rhinovirus: 28.1%, Asian influenza virus: 6.3%,
Adenovirus: 5.7%.


20

Table 3.30. Rates of autosome process between H. influenza and viruses causing RID according
to ages:
HI and
HI and influenza
viruses
virus type A

HI and RSV

HI and Rhinovirus

HI and other types
of viruses

Ages

Negativ
Negativ
Negativ
Negativ
Positive
Positive
Positive
Positive
e
e
e
e
< 12
126
6
121
11
115
17
125

7
months (30,9%) (18,2%) (30,7%) (23,4%) (31,4%) (22,7%) (31,5%) (15,9%)
12-23
175
17
171
21
148
44
168
24
months (42,9%) (51,5%) (43,4%) (44,7%) (40,5%) (58,6%) (42,3%) (54,6%)
24-<60
107
10
102
15
103
14
104
13
months (26,2%) (30,3%) (25,9%) (31,9%) (28,1%) (18,7%) (26,2%) (29,5%)
Total
408
33
394
47
366
75
397

44
(100,0) (100,0) (100,0) (100,0) (100,0) (100,0) (100,0) (100,0)
P
=0,308
=0,512
=0,015
=0,094
The rates of autosome process between H. influenzae and Rhinovirus was mainly at
the age of 12-23 months, accounting for 58.6%, these differences had statistical meanings
with p < 0.05. The rates of autosome process between H. influenzae with other viruses
causing RID in under 5-year-old children had no statistical meaning with p > 0.05.
Table 3.32. Rates of autosome process between H. influenzae and viruses causing RID according
to seasons:
HI and
HI and influenza
viruses
virus type A

HI and RSV

HI and Rhinovirus

HI and other types
of viruses
Negativ
e

Ages

Negativ

Negativ
Positive
Positive
e
e

Negativ
e

Dry
seasons
Rainy
seasons
Total

266
15
(65,2%) (45,5%)
142
18
(34,8%) (54,5%)
408
33
(100,0)
(100,0)
=0,023

231
50
(63,1%) (66,7%)

135
25
(36,9%) (33,3%)
366
75
(100,0)
(100,0)
=0,560

P

243
38
(61,7%) (80,9%)
151
9
(38,3%) (19,1%)
394
47
(100,0)
(100,0)
=0,010

Positive

Positive

251
30
(63,2%)

(68,2%)
146
14
(36,8%) (31,8%)
397
44
(100,0)
(100,0)
=0,516

The rate of autosome process between H. influenza and influenza virus type A was
popular in rainy seasons, accounting for 54.5 %, the differences had statistical meanings
with p < 0.05; the autosome process rate between H. influenzae and RSV was mainly in dry
seasons, making up 80.9 %, the differences had statistical meanings with p > 0.05.


21

3.3. Findings from determining relating factors of RID in under 5-year-old children:
3.3.1. Findings from determining origins causing RID according to ages

Figure 3.8. Rates of determining causal bacteria causing RI disease according to ages

The rates of S. pneumoniae causing RID was highly determined at the age of 12-23
months, accounting for 46.9 %, the differences had statistical meanings with P < 0,05; the
rate of H. influenzae was popular at the age of 12-23 months, accounting for 64.1%, the
differences had statistical meanings with P < 0.05; the rate of M.catarrhalis was popular at
the age of 12-23 months, accounting for 31.8%, the differences had statistical meanings
with P < 0.05
3.3.2. Findings from determining the origins causing RID according to gender

Table 3.37. Rates of determining origin viruses causing RI diseases according to gender:
Viruses Influenza virus type
RSV
A
Gender Negative Positive Negative Positive
147
24
130
41
Girls
(39,5%) (34,8%) (37,7%) (42,7%)
225
45
215
55
Boys
(60,5%) (65,2%) (62,3%) (57,3%)
372
69
345
96
Total
(100,0)
(100,0)
(100,0)
(100,0)
P
=0,285
=0,371

Rhinovirus

Adenovirus

Negative Positive
126
45
(39,7%) (36,3%)
191
79
(60,3%) (63,7%)
317
124
(100,0)
(100,0)
=0,503

Negative Positive
162
9
(38,9%) (36,0%)
254
16
(61,1%) (64,0%)
416
25
(100,0) (100,0)
=0,769

The rates of origin viruses causing RIDin under 5-year-old children determined

according to the gender had no statistical meaning with p > 0.05.
3.3.3. Findings from determining causes of RID according to seasons
Table 3.38. Rate of determining causes of RIDaccording to seasons:
Viruses Influenza virus type
RSV
A
Gender Negative Positive Negative Positive
Dry
246
35
203
78
season
(66,1%) (50,7%) (58,8%) (81,2%)
s
Rainy
126
34
142
18
season
(33,9%) (49,3%) (41,2%) (18,8%)
s
Total
372
69
345
96
(100,0)
(100,0)

(100,0)
(100,0)

Rhinovirus

Adenovirus

Negative

Positive

Negative

202
(63,7%)

79
(63,7%)

264
(63,5%)

17
(68,0%)

115
(36,3%)

45
(36,3%)

152
(36,5%)

8
(32,0%)

317
(100,0)

124
(100,0)

416
(100,0)

25
(100,0)


22
P

=0,015

=0,0001

=0,998

=0,647

The rates of influenza virus type A determined in the rainy seasons was 50.7 %, the
differences among seasons had statistical meanings with p < 0.05 %; the rate of RSV
determined in the dry seasons was 81.2 %, the differences among seasons had statistical
meanings with p < 0.05; the rates of Rhinovirus and Adenovirus differently determined had
no statistical meanings with p > 0.05
Chapter 4. DISCUSSION
4.1. Background restrictions of some bacteria causing RID in under 5-year-old
children:
S. pneumoniae is both the factors causing RID in under 5-year-old children and the
component of normal bacteria system. Therefore, experiments to find S. pneumoniae could
not ensure that it is the cause of the disease. My studies on three groups of children
included: pneumonia -X rays (+), bronchitis, and healthy. At concentration ≥ 107 CFU/1ml
of the medical waste, the difference of the rate of S. pneumoniae subdivided from 3 groups
of children had statistical meanings with p < 0.05. Of which, the inadequacy of the rate of S.
pneumoniae subdivided from the pneumonia-X rays (+) group and the healthy group was
4.8 times.
To conclude, at concentration ≥ 107 CFU/1ml of the medical waste, the difference of
the Rate of S. pneumoniae subdivided from 3 groups of children had statistical meanings
with p < 0.05. Of which, the inadequacy of the Rate of S. pneumoniae subdivided from the
pneumonia-X rays (+) group and the healthy group was 4.8 times. This can be considered
restriction to define children infected with RID.
The rates of healthy people catching Hib is from 1% to 5%, up to 10 % in some
regions. H. influenzae is both the factors causing RIDin under 5-year-old children and the
component of normal bacteria system. Therefore, experiments to find H. influenzae could
not ensure that it is the factor of the disease. My study on three groups of children included:
pneumonia -X rays (+), bronchitis, and healthy. At concentration ≥ 107 CFU/1ml of the
medical waste, the differences of the rates of H. influenzae subdivided from 3 groups of
children had the statistical meaning with p < 0.05. Of which, the inadequacy of the Rate of
S. H. influenzae subdivided from the bronchitis and healthy groups was 2.1 times.

To conclude: At concentration ≥ 107 CFU/1ml of the medical waste, the difference of
the rates of H. influenzae subdivided from 3 groups of children had the statistical meanings
with p < 0.05. Of which, the inadequacy of the rates of H. influenzae subdivided from the
bronchitis and healthy groups was only 2.1 times. Therefore, it cannot be concluded as the
restriction to define children infected with RID.
M. catarrhalis is both the factors causing RID in under 5-year-old children and the
component of normal bacteria system. Therefore, experiments to find M. catarrhalis could
not ensure that it is the factor of the disease. The Rate of having M. catarrhalis that we