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研究生:劉虹茗
研究生(外文):Luong Minh Hoa
論文名稱:越南北部高危險群感染H5N1之流行病學研究
論文名稱(外文):An Epidemiological Study of H5N1 Viral Infection among Different High Risk Groups from Three Northern Provinces of Vietnam
指導教授:陳宜民陳宜民引用關係
指導教授(外文):Yi-Ming Chen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:公共衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:85
中文關鍵詞:N/A
外文關鍵詞:H5N1 infectionEpidemiological StudyHigh risk groupVietnam
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The outbreaks of highly pathogen avian influenza H5N1 virus have occurred in many countries since 2003 Vietnam has many reports of human infection with this virus. However, the H5N1 virus epidemics in the communities and its transmission from poultry to human, or even directly human to human is unclear in Vietnam.
A total of 149 single sera samples were collected from poultry raiser, slaughterer and health care worker occupations living in 3 provinces of Northern Vietnam during September 2006 – March 2007, they were used for diagnosis of H5N1 infection by different assays including Haemagglutiation inhibition (HI), Micro-neutralization test (MN), H5N1 virus and H5 protein coated (ELISA) and H5-western blot (WB) which using H5-hemagglutinin as reactive antigens.
Our results indicate that 49.0 %, 16.1 %, 35.6 %, 40.3% and 34.2 % were positive in HI, MN, H5N1-ELISA, H5-ELISA and H5-WB, respectively. Comparisons of these serological assays with H5-WB test, we found that the H5-ELISA for detecting antibodies against H5N1 virus showed the highest specificity (87%) and sensitivity (68%). Concerning the risk factors associated with H5N1 infection, the following two risk factors were identified: 1. had contact history with sick or dead poultry in household or slaughter-house (p<0.05) and 2. direct contact with H5N1 patients (p< 0.05).
In summary, the assays developed in this study are useful for the serological diagnosis of H5N1 infection and direct contact with H5N1-infected poultry or patients is the most important risk factor for H5N1 infection.
CONTENTS
ABBREVIATIONS. .................................................................................................................. vi
ABSTRACT ............................................................................................................................... 1
CHAPTER 1: INTRODUCTION ........................................................................................... 2
1.1 Influenza A virus ............................................................................................................ 3
1.2 Avian influenza H5N1 subtype ...................................................................................... 3
1.3 Transmission of influenza A subtype H5N1 .................................................................. 4
1.4 H5N1 virus epidemic in the world ................................................................................. 5
1.5 H5N1 virus epidemic in Vietnam ................................................................................... 7
1.6 Study necessity and urgency .......................................................................................... 8
1.7 H5N1 Serologic testing methods .................................................................................... 9
1.8 Supposition of study ..................................................................................................... 10
1.9 Aims of the study .......................................................................................................... 10
CHAPTER 2: MATERIALS AND METHODS .................................................................. 12
2.1 Study design. ................................................................................................................ 13
2.2 Selection of study samples and objects. ....................................................................... 13
2.3 Sample size. .................................................................................................................. 13
2.4 Definition of study criteria ........................................................................................... 14
2.5 Hemagglutination Inhibition-HI assay ......................................................................... 14
2.6 Micro neutralization (MN) assay .................................................................................. 17
2.7 ELISA assay ................................................................................................................. 20
2.7.1 ELISA- H5N1 A/Vietnam/1203/04 strain .................................................................... 20
2.7.2 ELISA- H5 protein ....................................................................................................... 21
2.8 Western Blot assay. ...................................................................................................... 22
2.9 Statistical analysis ........................................................................................................ 23
CHAPTER 3: RESULTS ....................................................................................................... 24
3.1 Result of HI assay to detect antibody against avian influenza H5N1 in high risk of occupational group. .................................................................................................................. 25
3.2 Result of Micro-Neutralization assay to detect antibody against avian influenza H5N1 in high risk of occupational groups. ......................................................................................... 25
3.3 Result of ELISA assay to detect antibody against avian influenza H5N1 in high risk occupational groups .................................................................................................................. 26
3.3.1 Result of ELISA test using A/Vietnam/1203/04 H5N1 virus ...................................... 26
3.3.2 Result of ELISA test using highly purified baculovirus-expressed HA/H5 protein from A/Thailand/KAN-1/2004 virus ................................................................................................. 27
3.4 Result of Western Blot ................................................................................................. 27
3.4.1 Assessment of cross-reactive season flu vaccine against H5N1 influenza by using H5 specific Western blot assay....................................................................................................... 28
3.4.2 Pairwise comparisions of Influenza virus Hemagglutinin amino acid sequences ........ 28
3.4.3 Result of Western-Blot assay to detect antibody against avian influenza H5N1 in a high risk of occupational group ................................................................................................ 28
3.5 Sensitivities and Specificities of serologic tests – Correlation between serologic tests for H5 antibody. ........................................................................................................................ 29
3.5.1 Sensitivities and Specificities of serologic tests for H5 antibody ................................ 29
3.5.2 Correlation between serologic tests for H5 antibody. .................................................. 30 ii
3.6 Characteristic of participants and occupational risk factors for H5N1 virus infection. 30
3.6.1 Characteristic of participants ........................................................................................ 30
3.6.2 Risk factors for H5N1 virus infection by age group .................................................... 30
3.6.3 Risk factor for H5N1 virus infections by gender ......................................................... 31
3.6.4 Risk factors for H5N1 virus infection in health care worker ....................................... 31
3.6.5 Risk factors for H5N1 virus infection in poultry raiser ................................................ 32
3.6.6 Risk factors for H5N1 virus infection in slaughterer. .................................................. 32
3.7 Characteristics and exposures of ten participants for H5N1 serum positive by five tests 33
CHAPTER 4: DISCUSSION ................................................................................................. 34
CHAPTER 5: CONCLUSIONS ............................................................................................ 41
CHAPTER 6: REFERENCES .............................................................................................. 43
CHAPTER 7: TABLES AND FIGURES ............................................................................. 50
Table 1. Titer of serum samples reactive in Hemagglutination Inhibition test for anti-H5 antibody ................................................................................................................................... 51
Table 2. Number and percentage of occupation whose serum tested positive for anti-H5 antibody by Hemmaglutination inhibition ................................................................................ 52
Table 3. Titer of serum samples reactive in Micro-neutralization test for anti-H5 antibody ... 53
Table 4. Number and percentage of occupation whose serum tested positive for anti-H5 antibody by MN ........................................................................................................................ 54
Table 5. The cut off value determination in H5N1 virus coated ELISA .................................. 55
Table 6. Titer of serum samples reactive in H5N1 virus coated ELISA for anti-H5 antibody 56
Table 7. Number and percentage of occupation whose serum tested positive for anti-H5 antibody by ELISA ................................................................................................................... 57
Table 8. The cut off value determination in HA-H5 virus coated ELISA................................ 58
Table 9. Titer of serum samples reactive in HA-H5 protein coated ELISA for anti-H5 antibody ................................................................................................................................... 59
Table 10. Number and percentage of occupational people whose serum tested positive for anti-H5 antibody by ELISA ...................................................................................................... 60
Table 11. Titer of human serum samples reactive in the flu vaccination program by serological tests ........................................................................................................................ 61
Table 12: Pairwise comparisions of Influenza virus Hemagglutinin amino acid sequences .... 62
Table 12: Pairwise comparisions of Influenza virus Hemagglutinin amino acid sequences (cont) 63
Table 12: Pairwise comparisions of Influenza virus Hemagglutinin amino acid sequences (cont) 64
Table 13. Anti- H5N1 antibody evaluation by Western blot test using HA-H5 protein of human serum ............................................................................................................................ 66
Table 13. Anti- H5N1 antibody evaluation by Western blot test using HA-H5 protein of human serum (cont) .................................................................................................................. 67
Table 14. Number and percentage of occupation whose serum tested positive for anti-H5 antibody by Western Blot ......................................................................................................... 68
Table 15. Sensitivity and specificity of serologic tests for detection H5N1-antibody ............. 69
Table 16. Correlation between serologic tests for H5 antibody ............................................... 70
Table 17. Risk factor for H5 antibody among occupation group ............................................. 71
Table 18. Characteristic of 10 positive participants with H5N1 virus by five tests ................. 72
Table 19. Titer of samples reactive of ten H5N1 antibody positive participants with five tests 73
Table 20. Titer of samples reactive of negative control human serum samples for H5 antibody by five tests ............................................................................................................................... 74
Figure 1. Areas with confirmed human cases of H5N1avian influenza since 2003 ................. 75
Figure 2. The study areas of avian influenza H5N1 in Vietnam. ............................................. 76
Figure 3. Antibody reactivity of different groups of patients to Western blot assays using recombinant H5N1 hemagglutinin protein (arrow) produced from baculovirus-cell based system. ..................................................................................................................................... 77
Figure 4. Antibody reactivity of human serum samples tested positive and negative for H5 antibody by Western blot assays using recombinant H5N1 hemagglutinin protein (arrow) produced from baculovirus-cell based system.......................................................................... 78
Figure 4. Antibody reactivity of human serum samples tested positive and negative for H5 antibody by Western blot assays using recombinant H5N1 hemagglutinin protein (arrow) produced from baculovirus-cell based system (cont) ............................................................... 79
Figure 4. Antibody reactivity of human serum samples tested positive and negative for H5 antibody by Western blot assays using recombinant H5N1 hemagglutinin protein (arrow) produced from baculovirus-cell based system (cont) ............................................................... 80
Figure 5. Antibody reactivity of negative control of human serum samples who have lived in the temple of Vietnam for H5 antibody by Western blot assays using recombinant H5N1 hemagglutinin protein (arrow) produced from baculovirus-cell based system ........................ 81
ACKNOWLEDGEMENTS. .................................................................................................... 82
APPENDIX .............................................................................................................................. 83

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