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

Identifing the causes of ARI among children under 5 years old and the antibiotic sensitivity of some common bacteria.. The causes of ARI among children under 5 years old and the antibiot

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

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

TABLE OF ABBREVIATIONS

H influenzae Haemophilus influenzae

M catarrhalis Moraxella catarrhalis

RSV Respiratory Syncytial Virus

S pneumoniae Streptococcus pneumoniae

Acute respiratory infections (ARI) are common infectious diseases among chidren indeveloped and developing countries According to the statistics of World HealthOrganization (WHO), some 15 million children die each year globally, 95 percent of whomare in developing countries and 4 million of them died because of ARI Therefore, WHOconsiders the identification and prevention of the origin of ARI as one of their foundationalstratergies in order to improve children’s health

In Vietnam, the rate of ARI incidence and death are now high, which is ranked as thefirst rate of the acute infection diseases A wide variety of viruses, bacteria or parasites arethe origin of ARI In developed countries, ARI is almost caused by viruses, which accountsfor 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 identifythe fundamental limit in order to decide whether the bacteria are nomal ones or pathogenicones Although WHO has been attempted to prevent ARI in global scale, the rate of ARIincidence 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

STRUCTURE OF THE STUDY

This disseartation consists of 125 pages, exclusive of references and appendix There are 4chapters, 38 tables, 10 graps, 3 pictures and 125 domestic and international referencematerials The structure of the dissertation includes: 2 pages of Introduction, 35 pages ofLiterature 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

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 todevelop its structure and functions until the child is borned, and respiratory system startsworking However, since its structure and fuctions are not fully developed, child’srespiratory 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 lowerrespiratory tract The airway resistance of respiratory system is very strong, but theexpansion of chest is weak For respiratory tract disease, the mucosa has many bloodvessels, 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, theyencounter the system of respiratory protection including mucosal barrier, humoral factorsand 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% healthypeople carry aureus in theỉ nose, and this proportion is higher among people working in thehospital 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 usuallyhas Streptococcus group A

- Bacteria in the trachea and bronchi: due to the structural characteristics, physiology hasmucous 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

ARI among children under 5 years old is ranked at the hightest rate of incidence and deathworldwide The mortality rate of ARI in developing countries is 30-5- higher than that indeveloped countries Study shows that: in developed countries, the origin of ARI is almostviruses, which accounts for 80-90 percent In developing contries, the origin of ARI isbacteria 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 ofARI among children is 65,8%, which is the highest rate among the common disease in thecommunity

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 alot In young children, some symptoms are high fever, convulsions, inflammation of thestomach - 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 bestspecimens for influenza diagnosis are nasal throat swabs combined with mouth and throatswabs

- The specimen for serological diagnosis: blood in the acute phase is taken as soon aspossible After 14-28 days, blood in the recovery phase is taken Serum is stored at -200Cuntil it is tested

negative-+ 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:

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 acold, 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 cellrecptors in order to find giant cell In addition, diagnostic methods are also determined byimmunofluorescence, PCR

1.2.2.3 Adenovirus:

Pathogenic role: viruses that cause acute infection have some characters such as: a shortincubation period, prolonged excretion virus, mild disease However, pneumonia in youngchildren 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 causedisease

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 aredamaged more than 25%, PCR or immunofluorescence is conducted to identify theAdenovirus

1.2.2.4 Rhinovirus:

1.2.2.4 Rhinovirus:

Role pathogens: rhinovirus enters the body through inhaling the particles of respiratorysecretions of an infected person or indirectly through hands, hand towels, furniture, toys ofinfected children’s respiratory secretions containing viruses The viruses enter the bodythrough the upper respiratory tract and are often limited here

Diagnostic from laboratories: the laboratory diagnosis relies on virus isolation from nasaland throat secretions, in culturing cells Use PCR techniques to identify specific gene of thevirus

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 causerespiratory 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:

* Gram stain: nose and throat swabs are used to identify the causes Because many types ofbacteria are mixed toghether, if bacteria are identified, the bacteria may belong to thenormal 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, alphahemolytic

- The nature of bacteria: diplococcus in the shape of a candle or a pair of glasses whichcatch color Gram (+) and is sensitivity to optochin (diameter of sterile cirlce ≥ 14 mm) or isless 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 besoluble 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) Nontypeableoften 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 causehemolytic

- 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

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 5years 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 antibioticresistance 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 atdifferent levels and are manifested in the difference in diameters around the sterile circular

of antibiotic paper, which contain determined attenuation when the contact between thebacteria with antibiotics occurs

* Purpose: Study on the antibiotic sensitivitive of bacteria helps clinical direction have righttreatment with specific antibiotics, and help contribute to evaluation of the situation of drug-resistance 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 certainconcentration, it will inhibit the growth of bacteria and it can be determined by the humaneyes

* 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 350under 5-year-old healthy children in Vinh Thanh and Vinh Phuong Commune, Nha TrangCity

- Diseased group includes 441 clinical specimens of nasopharyngeal juice taken from 441under 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 ofnasopharyngeal 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 InhibitoryConcentration (MIC) method

- PCR was used to identify S pneumoniae and H Influenzae which contained protestinggenes

- 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

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 Theclinical specimens of nasopharyngeal juice taken from healthy children and under 5-year-old children sufferring RID based on PCR system; This experiment will be applied in thelaboratories of diagnosis of bacterial respiratory diseases in Vietnam Multi-primer is todetect such three types of bacteria as S pneumoniae, H influenzae, M catarrhalis and threequanlitative PCR reations based on the principle of Syber Green that is used to quantifyeach 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 year-old healthy children in Vinh Thanh and Vinh Phuong Commune, Nha Trang City and

5-441 clinical specimens of nasopharyngeal juice taken from 5-441 under 5-year-old childrensufferring RID in Pediatrics of Khanh Hoa Hospital Samples were taken according to theroutine 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 aretested

* 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, theDNA is determined by the concentration and purity of its optical gauges Eppendorf AG ofGermany The DNA samples are preserved at minor 300C

 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 formulti-bait PCR are reevaluated by single-bait PCR The positive clinical specimens are

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 107CFU 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 respiratorydiseases through Multiplex RT-PCR and Heminested PCR method

- Set of biological products to separate ARN: QIAamp viral RNA Mini Kit, 250 preps, Cat

Use four multiplex PCR reactions to detect 13 viruses causing respiratory diseases inthe clinical specimens: Rhinovirus, Respiratory Syncytial Virus, flu virus A and B, flu virustube 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 ethidiumbromide ( 10 1/100ml of agarose fluid), voltage of 100V in 30 minutes Use the molecularweight scale 100bp The AND bands can be observed and shot under the UV ray

 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 nopresence 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 culturingenvironment

- Principles: The concentration of antibiotics increase in the culturing environment, at acertain 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 andmade pure Choose randomly 89 races of S pneumonia and 37 races of H influenza to testMIC and PCR method to determine the gene of drug resistance

- Steps: Following the regulations of World Health Organization

- 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 whichthe bacteria are inhibited to develop MIC result is compared with antibiotic boundaryconcentration 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 byusing 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 therelationship 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 researchsubject were clearly explained the research objectives and had agreement in participation.The private information was absolutely confidential The fee of experiments for the clinicalspecimens of nasopharyngeal juice is freely covered all The research subjects are lookedafter and treated as other patients The experiment results are used for diagnosis, preventionand 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:

(n)

Rates (%) having S pneumoniae

 105CFU/1ml

106CFU/1ml

 107CFU/1ml

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 statisticalmeanings 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.

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:

(n)

Rates (%) having influenzae

 105CFU/1ml

106CFU/1ml

 107CFU/1ml

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.