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

1 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 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: Assoc.Prof., Dr ĐẶNG ĐỨC ANH 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 year 2012 This doctoral thesis could be found at: - The National Library - The National Institute of Hygiene and Epidemiology Library 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 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 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 Adenovirus In developing countries, the origin of ARI is bacteria, accounting for 75 percent The common bacteria are S pneumoniae, H influenzae 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 years old in Nha Trang City, 2009” OBJECTIVES OF THE STUDY Identifing the fundamental limits of some causes of ARI among children under years old Identifing the causes of ARI among children under years old and the antibiotic sensitivity of some common bacteria Investigate several factors related to the causes of ARI among children under years old RESEARCH SIGNICANCE 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 Determine the distribution ratio of bacteria, viruses that may cause disease in healthy children and children under the age of who suffer from ARI Update information on the antibiotic sensitivity of some certain bacteria causing ARI in children and point out some factors related to ARI in children STRUCTURE OF THE STUDY This disseartation consists of 125 pages, exclusive of references and appendix There are chapters, 38 tables, 10 graps, pictures and 125 domestic and international reference materials The structure of the dissertation includes: pages of Introduction, 35 pages of Literature review, 26 pages of Methodology, 31 pages of Findings, 28 pages of Discussion, pages of Conclusion, page of Implications and 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 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 years old and the antibiotic sensitivity of some common bacteria 1.2.1.1 The real situation of researching on ARI among children under years old 1.2.1.1 Research on ARI among children under years old in the world ARI among children under 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 years old in Vietnam: In Vietnam, the rate of ARI incidence and death is mainly among children under 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 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: Pathogenic role: RSV is spread through contact with droplet secretions containing viruses Children from weeks to 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 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 cells which is cell-specific receptor for Adenovirus The plastic blade used to transplant Hep 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 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: * 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 128 >128 >128 10,1 5,6 84,3 Chloramphenicol 0,5-32 65,2 23,6 11,2 Co - trimoxazole 4->128 64 >128 5,6 94,4 Clindamycin 0,004->128 128 >128 20,2 79,8 10 Ofloxacin 0,5-4 2 92,1 7,9 11 Tetracycline 0,125-128 32 64 11,2 88,8 12 Rifampicin 0,032-0,25 0,125 0,25 100 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 (%) pbp sudden mutation 139 91,5 pbp sudden mutation 11 7,2 pbp sudden mutation 0,7 pbp normal 0,7 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 pbp, accounting for 91.5%, 11 types had pbp, making up 7.2%, type had pbp, namely 0.7%, typ had 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 (%) Negative 3,3 gene ermB 93 61,2 gene mefA 3,3 gene ermB+mefA 49 32,2 Total 152 100,0 Of 152 types of S pneumoniae causing RID that were subdivided, there are types that did not have the antibiotic resisting macrolide gene, accounting for 3.3%, 93 types had ermB gene, accounting for 61.2%, types had 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 Ampicillin 0.125 - 64 >64 >64 40,5 59,5 Amoxycillin/Cla 0.125 - 97,3 2,7 Ampicillin/Sul 0.063 - 16 0,5 78,4 21,6 Cefuroxime 0.5 - 64 32 63,8 16,7 19,4 Cfotaxime 0.008 - 0,032 0,5 100 0 Ceftriaxone 0.004 - 0.25 0,008 0,125 100 0 Ceftazidime 0.063 - 0,125 100 0 Cefepime 0.063 - 0,125 100 0 Azithromycin 1,0-4,0 2 100 0 10 Clazithromycin 2,0-16 91,2 8,8 11 Chloramphenicol 0,25-16 16 56,8 43,2 12 Tetracycline 0,25-32 0,5 16 62 38 13 Ciprofloxacin 0,008-0,016 0,016 0,016 100 0 14 Co - trimoxazole 0,5->128 >128 >128 13,5 86,5 15 Rifampicin 0,25-1 0,5 0,5 100 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 (%) Negative 53 51 TEM – 51 49 ROB – 0 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 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 < 12 126 121 11 115 17 125 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- 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 (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 (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%) (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 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 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 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 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 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 23 determined was so high, making up 58.0 % 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 M catarrhalis subdivided from groups of children had the statistical meaning with p < 0.05, of which, the inadequacy of the rates of M catarrhalis subdivided from the bronchitis and healthy groups was 2.1 times To conclude: At concentration ≥ 107 CFU/1ml of the medical waste, the difference sof the rates of M catarrhalis subdivided from groups of children had the statistical meanings with p < 0.05 Of which, the inadequacy of the rates of M catarrhalis 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 4.2 The cause of RID in under 5-year-old children 4.2.1 Causal bacteria of RID in under 5-year-old children * The rates of S pneumoniae determined in under 5-year-old healthy children The rate of S pneumoniae that we determined in under 5-year-old healthy children was 50.3 % In comparison with that of some inland authors, this rate is far higher, specifically: Phạm Văn Ca and Lê Đăng Hà (2003) at Vị Xuyên 10,7% and Vân Đồn 15% In comparison with that of foreign authors, my findings were higher For example, findings from Morrissey and his partners’ study in HongKong on 1455 under 5-year-old children showed the Rate of S pneumoniae determined was 36%; Revai K and partners examined over 1214 healthy children in the USA and got the Rate of 44% of S pneumonia The rates of S pneumoniae determined in under 5-year-old children remained different since Vietnam is a developing countries, the environment is not good, the nurturing and hygiene conditions are not appropriate Therefore, they are contributing to increase the rate of catching S pneumoniae’s diseases * The rates of S pneumoniae determined in under 5-year-old children catching RI disease From 441 medical waste samples (nose throat juices of under 5-year-old children catching RI disease), the rate of S pneumonia determined was 38.8% Masuda K and his partners have investigated on 156 medical waste samples (nose throat juices of 156 children patients at the age of to 59 months catching RI disease) and their findings showed that the Rate of S pneumoniae subdivided was 60.3% Đặng Đức Anh and his partners have made an investigation to determine the causal bacteria of RI in National Children Hospital in the period of 2002-2003 The findings from that research figured out that the Rate of S pneumoniae subdivided was 48% Our findings, compared with that of inland and overseas authors, had lower rate of S pneumonia The differences are due to the fact that the researches have been done in different time, places and by different experimenting methods 24 * The rates of H influenzae determined in under 5-year-old children catching RI disease: The rate of H.influenzae that we determined in under 5-year-old healthy children was 31.4 % In comparison with that of previous studies of inland authors such as Phạm Văn Ca and Lê Đăng Hà (2003) at Vị Xuyên 24%, at Vân Đồn 14,7%, the rate of H.influenzae of our research remains higher The rate of H influenzae virus in respiratory vessels changes in ages, specially the healthy people: 20 - 40 %, healthy children: 75 - 80% The rate of healthy people catching Hib varies from to 5%, up to 10% in some regions However, in some kindergartens where there are children catching meningitis due to Hib, this rate ups to 70 % In our research, the H.influenzae determined in healthy children is 31.4 % * The rates of H influenzae determined in under 5-year-old children catching RI disease From 441 medical waste samples (nose throat juices of under 5-year-old children catching RI disease), the determined rate of H influenzae was 51,5% Masuda K and his partners have made an investigation on 156 medical waste samples (nose throat juices of 156 children patients at the age of to 59 months catching RI disease) and got the rate of H influenzae of 53,2% Đặng Đức Anh and his partners have investigated to determine the causal bacteria of RI in National Children Hospital in the period of 2002-2003 The findings from that research figured out that the Rate of H influenzae subdivided was 44% My findings, compared with those of foreign authors, show that the rate of H influenzae subdivided was much lower In comparison with those of inland authors, the rate of H influenzae determined was higher Our studies on group of children at the age of 12-23 months showed that the rate of H influenzae was the highest with 54.2% There were the differences since the amount of antibody that the mothers supply decreases gradually from the 6th month; on the other hand, the children’s immune systems, especially the antibody juices and the amount of IgA secreted in the surface of respiratory vessels’ mucous membrane remain incomplete and low The existence of it cover is one of the factor for H influenzae type b protest against phagocyte In my studies, most of the determined types of H influenzae from the nose-throat juices had no cover, accounting for 96.1 %, the number of types that had cover were 3.9 % All of them were H.influenzae type b * The rates of M catarrhalis determined in under 5-year-old healthy children: From the previously inland and oversea studies, there were 75% of the children having M catarrhalis in their respriratory vessels; this rate in adults was just to 3% * The rates of M catarrhalis determined in under 5-year-old children catching RI disease: From 441 medical waste samples (nose throat juices of under 5-year-old children catching RI disease), the rate of M catarrhalis determined was 31.5% Masuda K and his partners have investigated on 156 medical waste samples (nose throat juices of 156 children patients at the age of to 59 months catching RI disease) and their findings showed that the rate of M catarrhalis subdivided was 34.6% Đặng Đức Anh and his partners have made an 25 investigation to determine the causal bacteria of RI in National Children Hospital in the period of 2002-2003 The findings from that research figured out that the rate of M catarrhalis subdivided was 25.5% Our findings, compared with that of Masuda K., had lower rate of M catarrhalis; however, in comparison with those of inland authors, the rate of M catarrhalis was higher My studies have pointed out that the rate of M catarrhalis determined in group of children at the age of 12-23 months accounted for 31.8% The differences in determining M catarrhalis among ages had statistical meanings with p < 0.05 4.2.2 The level of sensibility to antibiotic of various types of bacteria causing RIDin under 5-year-old children: * The level of sensibility to antibiotic of S.pneumoniae: Our study was on 89 types of S pneumoniae which were subdivided and expreminented with antibiotic by MIC techniques The results showed that S pneumoniae was totally sensitive to rifampicin, 92.1% to vofloxacin and 85.4% to amoxycillin/clavulanate To penicillin G, S pneumoniae was 7.9% sensitive, had only 9.0 % for real resistance, but had 83.1% for reducing sensibility The levels of resisting other antibiotic remained high: co-trimoxazole: 94.4% and erythromycin: 84,3% The level of sensibility to co-trimoxazole was only 5.6% These results were much lower than those of the program on supervising antibiotic resistance of bacteria in 2002 (42.5%) and 2003 (85,4%), Nguyễn Thị Thanh Hà (2000, 24%) On the other hand, these results were higher than that of an investigation of ASTS program at Hue National Hospital in 2003 (0%) There have been differences because the researches were done at different times and places Furthermore, although co-trimoxazole had large acting scale, it oly had bacteria resisting functions Therefore, after long time widely used, the decrease of level of sensibility is reasonable Wide and wrong application of antibiotic has increased the antibiotic resisting of S.pneumoniae The resisting mechanism: Because S pneumonia changes the target structure so it can resist penicillin, which leads to decreasing in percepting penicillin attaching protein Of pbp of S pneumonia, 1a, 2b, 2x are those pbp that are relating to anti penicillin Our studies’ results indicated that of 152 types of S pneumoniae subdivided, there have 139 types that had pbp sudden mutation, accounting for 91.5%; 11 types had pbp sudden mutation, namely 7.2%; type had pbp sudden mutation, making up 0.7% and type had normal pbp, which was 0.7% S pneumoniae resists macrolide mainly with mechanism: methylysation of bacterial ribosome (due to ermB gene) or forming the biological pump, increasing antibiotic emission (by mefA gene) Our studies showed that of 152 types of S pneumoniae subdivided, there were types that did not have macrolide’s antibiotic resisting genes, making up 3.3%; 93 types had ermB genes, accounting for 61.2%; types had mefA genes, making up 3.3% and 49 types had both ermB and mefA genes, accountung for 32,2% 26 * In conlusion, S pneumoniae causing RID in under 5-year-old children are totally sensitive to rifampicin; the level of sensibility to ofloxacin is 92.1% However, the level of resisting co-trimoxazole is 94.4% and to erythromycin is 84.3% S pneumoniae resists penicillin because of sudden mutation of pbp and it resists macrolide because it have ermB, mefA or both ermB and mefA gene * Levels of sensibility to antibiotic of H influenzae: Phan Lê Thanh Hương’s research on the causal bacteria and level of sensibility to antibiotic in RID of children in 2002-2003 at National Children hospital showed that H influenzae resists ampicillin at the level of 35%, resists co-trimoxazole at the level of 82,5% Our studies were on 37 types of H influenzae that were subdivided, made an experiment on with antibiotics using MIC technique The results showed that H.influenzae was totally sensitive to Cefotaxime, ceftriazone, azithromycin but resisted co – trimoxazole at the level of 86.5%, and ampicillin at the level of 59.5% These results, in comparison with those of other inland authors, are higher These are due to incorrect way of using antibiotics and antibiotic abusing, which leads to the dramatic increase of medicine-resisting of H influenzae H influenzae resists antibiotic type β-lactam because its bacteria produce β-lactamase enzyme The production of β-lactamase in any type of H influenzae bacteria is usually made by two types of genes: blaTEM-1 (TEM-1 β-lactamase) and blaROB-1 (ROB-1 βlactamase) In our studies, of 104 type of H Influenzae subdivided, there were 53 type that did not have gene of medicine resisting type β-lactam, accounting for 51,0%, 51 types of H influenzae had gene of medicine resisting type β-lactam blaTEM -1, no type had gene of medicine resisting type β-lactam blaROB-1 In conclusion, any types of H influenzae causing RID in under 5-year-old children that we subdivided are totally sensitive to antibiotics such as cefortaxime, ceftriaxone and azithromycin but they resisit co-trimoxazole at the level of 86.5% ampicillin at the level of 59,5% H influenzae resist β-lactam because it had blaTEM-1 gene, accounting for 49%, no type had blaROB-1 gene 4.2.3 Causal viruses of RID in under 5-year-old children: Our studies on 441 medical waste samples (nose-throat juices of under 5-year-old children) pointed out that 307 medical wastes were determined to include viruses, of which influenza virus type A accounts for 15.6 %, Rhinovirus: 28.1%, Adenovirus: 5.7%, other types of viruses: 13.6% Straliotto S.M and his partners made an investigation on nose-throat juices of 862 under-5-year-old children who were diagnosed to catch RID By the fluorescent immune method, they determined 316 medical waste samples to have viruses, accounting for 36.6% Of those viruses, respiratory vessel plasmodium viruses accounted for 26.2 %, Adenovirus 27 made 6.0%, influenza virus were of 2.9%, half-influenza viruses made up 1.5% Đặng Đức Anh and his partners made an experiment on 198 medical waste samples (nose-throat juices of under 5-year-old catching RID at Department of Children Respiratory of Xanh Pon hospital) Their study figured out that Influenza viruses type A accounted for 1.5%, Adenovirus made up 2.5%, Coxsackievirus B was of 4.1% 4.3 Some factors relating to causes of RID in under 5-year-old children according to ages: 4.3.1 Causes of RID according to ages: In our studies, the under 12-month-old babies were determined to have some main bacteria causing RID such as S pneumoniae (accounting for 31.8%), H influenzae (34.8%), M catarrhalis (31,1%) For the 12-23-month-old babies, the rates of bacteria determined increase, for example the rate of S pneumoniae is up to 46.9%, H influenzae (64.1%) and M catarrhalis (31.8%) The differences of the rates of S pneumoniae, H influenzae and M catarrhalis determined according to ages have statistical meanings with P < 0.05 The study on the causal bacteria of RID in developing countries showed that S pneumoniae, M catarrhalis and H influenzae are the main factors causing the disease Our studies on 441 medical waste samples (nose-throat juices of under 5-year-old catching RID) used PCR technique to determine which viruses cause the disease Its results determined according to ages had no statistical meanings with p > 0.05 4.3.2 Causes of RID in under 5-year-old children according to gender: Any five-year-old children have no differences of immune system in comparison with that of others Therefore, the possibilities of catching microorganism of boys and girls are equal Many studies all over the world as well as in Vietnam on the rate of bacteria subdivided in under 5-year-old children have proved that there are no differences between boys and girls Our studies showed that the rate of S pneumoniae, H influenzae and M catarrhalis determined according to gender were different, which had no statistical meanings with p > 0.05 The causes of RID are viruses, which we determined from different genders have no statistical meanings with p > 0,05 4.3.3 Causes of RIP in under 5-year-old children according to seasons: Chidren can have S pneumoniae, H influenzae, M catarrhalis in their respiratory vessel at any time of a year However, the rates of those bacteria change monthly Cold weather is the favourable condition for spreading S pneumoniae, H influenzae and M catarrhalis Our studies were carried out at Nha Trang cities, whether there are two different reasons: dry season and rainy season The rate of bacteria causing RID determined according to seasons had no statistical meanings with p > 0,05 28 Viruses causing RID in under 5-year-old children that we determined according to seasons were of influenza virus type A and respiratory vessel plasmodium viruses The differences had no statistical meanings with p < 0,05 4.3.4 The autosome process of causal bacteria of RID in under 5-year-old children: Our studies pointed out that there were some autosome process processes of bacteria in one medical waste, in which the autosome process of S pneumoniae and H influenzae was of 36.5%, S pneumoniae and M catarrhalis was of 29.9%, M catarrhalis and H influenzae was of 33.6% Determining correctly the rate of autosome processes of two kinds of bacteria benefits the treatment process because they are relating to chosing any kind of antibiotics that have effect on two kinds of the bacteria Our studies also showed that the rate of autosome process of bacteria causing RID is popular with children at the age of 1223 months The different autosome processes according to ages have no statistical meaning with p < 0,05 The different autosome processes of bacteria according to gender and seasons that we determined have no statistical meanings with p > 0.05 The results of experimenting 441 medical waste samples (nose-throat juices of under 5-year-old children catching RID) indicated that the rate of influenza virus type A was 15.6 %, respiratory vessel plasmodium viruses: 21.8%, Rhinovirus: 28.1%, Adenovirus: 5.7%, other types of viruses: 13.6% We also determined the autosome process of bacteria and viruses in one medical waste Determining correctly the rate of autosome processes of bacteria and viruses is important for choosing the appropriate techniques of treatment The different rates of autosome process of H influenzae and Rhinovirus according to groups of ages have statistical meanings with p < 0.05 The different autosome processes of H influenzae and influenza virus type A, of H influenzae and respiratory vessel plasmodium viruses according to seasons have statistical meaning with p < 0.05 CONCLUSION Background restriction: Background restriction of S pneumoniae is ≥ 107 CFU/1ml of medical waste of nosethroat juice (p < 0.05; the uneven rate of group of bronchitis chidren and healthy children is 4.8 times) The causes of RID in under 5-year-old children: * The rate of bacteria subdivided is 74.4%; of which S pneumoniae accounts for 38.8%; H influenzae: 51,5% (no-cover H influenzae makes up 96.1%, Hib:3,9%) and M catarrhalis accounts for 31.5% * The rate of viruses subdivided is 69.6%; of which influenza virus type A accounts for 15.6%, respiratory vessel plasmodium viruses: 21.8%, Rhinovirus: 28.1%, Adenovirus: 5.7% * The level of sensibility to antibiotics: 29 - S pneumoniae is sensitive to rifampicin 100%, ofloxacin 92.1%; resists co-trimoxazole 94.4%, tetracycline 88.8% and erythromycin 84.3% S pneumoniae resists penicillin because of sudden mutation of pbp and resists macrolide because it has ermB and mefA genes - H influenzae is totally sensitive to these antibiotics: cefortaxime, ceftriaxone, ceftazidime, azithromycin rifampicin and resists co-trimoxazole 86.5%, ampicillin 59.5% and chloramphenicol 43.2% H.influenzae resists β-lactam because it has blaTEM-1 gene and no blaROB-1 gene Factors relating to RID in under 5-year-old children: - Bacteria: the different rates of S pneumoniae, H influenzae M catarrhalis subdivided according to ages have no statistical meanings with p < 0.05 The rates of S pneumoniae, H influenzae M catarrhalis subdivided according to seasons have statistical meanings p > 0.05 The different rates of autosome processes of S pneumoniae and H influenzae according to ages have statistical meanings with p < 0.05 - Viruses: the rates of viruses subdivided according to ages and gender have no statistical meanings with p > 0.05 The differences between influenza virus type A and respiratory vessel plasmodium viruses have statistical meanings with p < 0,05 The different autosome processes of H influenzae and Rhinovirus according to ages have statistical meanings with p < 0.05; the differences of autosome processes of H influenzae and influenza virus type A A, H influenzae and respiratory vessel plasmodium viruses according to seasons have statistical meanings with < 0,05 IMPLICATIONS S pneumonia and H influenzae are the two main factors causing RID in children Therefore, it is needed to give injections of vaccines which are made of pneumococcus and Hib plus vaccines to children after months after they are born The rates of S pneumonia and H influenzae resisting antibiotics such as β – lactam and microlide have increased dramatically These are widely used antibiotics for treating RID Thus, there should be a supervision of antibiotic resistance and a plan of choosing alternative antibiotics 30 REFERENCE OF SCIENTIFIC RESEARCHES Vũ Văn Thành and Đặng Đức Anh (2010), “Causal bacteria of respiratory infection disease in under 5-year-old children at Nha Trang”, Journal of Preventive Medicine, Vol XX, No (113), pp 94-98 Vũ Văn Thành, Lay Mint Yoshida, Đặng Đức Anh and Phan Lê Thanh Hương (2011), Causal bacteria of respiratory infection disease in under 5-year-old children at Nha Trang in 2009” Journal of Preventive Medicine, Vol XXI, No (126), pp 95-100 ... respiratory infections among children under years old in Nha Trang City, 2009? ?? OBJECTIVES OF THE STUDY Identifing the fundamental limits of some causes of ARI among children under years old Identifing the. .. 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... be concluded as the restriction to define children infected with RID 4.2 The cause of RID in under 5- year -old children 4.2.1 Causal bacteria of RID in under 5- year -old children * The rates of