家禽及其暴露人群甲型流感病毒感染的流行病学调查研究

发布时间：2018-06-07 04:57

本文选题：甲型流感病毒 + 禽流感病毒　；参考：《中国人民解放军军事医学科学院》2014年博士论文

【摘要】：人感染禽流感病毒（avian influenza viruses, AIVs）的事件近年来报道愈加频繁。自从禽流感病毒H5N1于1997年被报道能够感染并致人死亡以来，已在15个国家造成658人感染，病死率近60%；禽流感病毒H7N7也于2003年在荷兰引起1人死亡；其他禽流感病毒如H9N2、H7N3等感染人事件在香港、加拿大等地也有报道。2013年初，禽流感病毒H7N9人感染病例又在我国东南部出现，截止2014年2月28日H7N9已经造成375人感染，115人死亡。因此，我国禽间AIVs感染及人群AIVs感染状况是备受关注的科学问题。进行H7N9禽流感病毒的现场调查，检测方法是关键一环。为了选择能够应用于现场H7N9禽流感病毒的快速、高效的检测方法，我们对5种用于检测人甲型流感感染的商品化试剂盒（Life Technologies VetMAXTM-Gold SIV DetectionKit、Quidel Molecular Influenza A+B assay、Remel Xpect Flu AB assay、QuidelQuickvue influenza assay以及Quidel Sofia Influenza A+B assay）进行了评价。首先从江苏省无锡市和南京市采集的健康活禽肛拭子样本提取RNA，用世界卫生组织（World Health Organization，WHO）推荐的实时荧光定量PCR（qReal-timePCR，qRT-PCR）influenza A assay和H7N9assay进行甲型流感病毒和禽流感病毒H7N9检测，筛选出27个H7N9阳性标本、70个H7N9及甲型流感阴性标本。然后对这97个标本使用上述5种试剂盒进行盲法检测以及WHO qRT-PCR H7N9assay的复核。检测时，根据试剂盒的不同试验原理，核酸分子检测方法每种重复3次，抗原检测方法每种重复2次。最后以WHO qRT-PCR H7N9assay作金标准，计算这几种方法的敏感度和特异度及其可信区间（confidence interval，CI）。结果：（1）WHO qRT-PCR H7N9assay第一轮和第二轮结果有很好的一致性。（2）Quidel Quickvue influenza assay对97份标本检测结果全部显示阴性。其他四种试验方法均有很好的特异度（96-100%）。（3）Life Technologies VetMAXTM-GoldSIV Detection Kit特异度最高，为100%（95%CI0.87-1.00），假阳性率4%（95%CI1-12%）。Quidel Molecular Influenza A+B assay敏感度较高，达85%（95%CI66-96%），假阳性率3%（95%CI0-10%）。为掌握AIVs在无锡地区家禽中的感染状况及其遗传进化特征，我们于2013年7月-12月每月在无锡市活禽交易市场和家禽养殖场的家禽中采集活禽及其相关环境拭子标本，以拭子标本提取的RNA为模板，运用WHO qRT-PCR influenzaA assay对样品进行甲型流感病毒筛查，对筛查阳性标本采用一步法反转录PCR进行流感病毒全基因组PCR扩增以及基因克隆和测序。测序所得的序列进行系统发育分析，同时对甲型流感病毒阳性标本进行MDCK细胞及鸡胚分离培养和鉴定。结果：（1）共采集标本305份，甲型流感病毒阳性率为28.5%（87/305）。家禽交易市场样本甲型流感病毒阳性率高于养殖场（31.9%vs.10.4%，χ2=9.16，P=0.002）；（2）共有6个样本可进行分型，分别为H9N2（3个），H5N8（1个），H11N2（1个）和H5N1（1个）。系统发育分析表明H5N8和H11N2有不同程度的重组。（3）共分离到3株禽流感病毒：A/Chicken/1/Wuxi/2013(H9N2)、A/Chicken/2/Wuxi/2013(H9N2)和A/Chicken/1/Wuxi/2013(H5N1)。禽间AIVs感染情况复杂多样，而禽类饲养、加工等职业暴露人群是感染AIVs的高危人群。除了感染AIVs发病甚至死亡之外，他们当中还存在诸如H5、H7、H9、H11等亚型AIVs的隐性感染者。猪能够感染猪流感病毒（swine influenzavirus，SIV），也能感染多种禽源和人源流感病毒，在“禽-猪-人”的种间传播链中扮演着流感病毒中间宿主及多重宿主的作用。研究已证明猪可感染禽流感病毒H5N1和H9N2。广东、福建、浙江地区约1%的猪职业暴露人群中有H9N2感染。因此，为了解禽和猪职业暴露人群甲型流感病毒的感染状况，通过分析感染与一般人口社会学情况和流行病学特征的关系，确定感染AIVs的危险因素，为人感染AIVs的防治策略提供依据，我们在无锡地区禽（猪）养殖场、屠宰场、市场和散养户集中地的暴露人群中开展了甲型流感病毒血清学现况调查。选择了禽职业暴露人群498名，猪职业暴露人群608名和无职业暴露的对照人群165名，共1271名研究对象。通过血凝抑制（hemagglutination inhibition, HI）试验和微量中和（microneutralization，MN）试验对研究对象血清标本进行了人甲型季节性流感H1N1（seasonal influenza A/H1N1，sH1N1）、sH3N2，及禽流感病毒H5N1、H9N2和H7N1血清抗体的检测。结果：（1）禽类职业暴露、猪职业暴露和对照人群的sH1N1HI抗体阳性率分别为17.7%（88/498），10.9%（66/608）和27.3%（45/165），三组sH1N1HI抗体阳性率差异有统计学意义（χ2=29.00，df=2，P=0.000）。sH3N2HI抗体阳性率分别为17.3%（86/498），6.4%（39/608）和8.5%（14/165），三组sH3N2HI抗体阳性率差异有统计学差异（χ2=34.29，df=2，P=0.000）。（2）H5N1在禽类职业暴露人群当中MN滴度≥1:80和≥1:160的分别占14.6%和8.4%，在猪职业暴露人群分别占0.2%和0.2%，对照人群中只有1名（0.6%）对象H5N1MN滴度为1:80，没有更高滴度的对象。多因素logistic回归分析表明，工作场所为市场的禽类职业暴露人员（OR=57.3，95%CI7.0-466.5）、慢性呼吸道疾病（OR=12.8，95%CI1.6-102.5）以及年龄40-60岁（OR=3.1，95%CI1.5-6.6）为H5N1MN滴度≥1:80的危险因素。（3）对H5N1MN滴度≥1:80的76份血清再做HI试验，最终按WHO MN滴度≥1:80且HI滴度≥1:160的确诊病例标准判定有3名H5N1感染者，为2名市场卖禽人员和1名规模化养鸡场工人，占禽暴露人群的0.6%（3/498），这3人中有2人否认近一年内有流感样症状。（4）H9N2试验结果有5名研究对象MN滴度≥1:10，其中2名是市场卖禽人员，2名是市场卖猪肉人员，1名是猪屠宰场工人。H9N2在禽职业暴露人群和猪暴露人群的MN抗体阳性率分别为0.4%（2/498）和0.5%（3/608）。对照人群中未发现H9N2MN抗体阳性者。（5）H7N1HI试验结果未发现抗体滴度≥1:10者。 H7N9禽流感病毒2013年初在中国东南部出现人感染病例，并在短期内呈迅速增长态势。活禽及相关环境（包括活禽市场）暴露被认为是感染H7N9的首要危险因素。在H7N9疫情延续期间，禽暴露人群是否有隐性感染？活禽市场的活禽从业人员和非活禽从业人员，以及活禽市场以外的禽类从业人员相比，感染的风险是否相同？感染与社会人口学、基础疾病、职业行为等因素又有什么样的关系？基于这些科学问题，我们在无锡地区禽职业暴露人群中开展了H7N9的血清学现况调查，共纳入对象1588名，，包括1178名活禽市场从业人员（300名活禽从业人员和878名非活禽从业人员），245名活禽市场以外的活禽从业人员和165名当地健康居民。实验方法选择火鸡血HI检测A/Anhui/1/2013(H7N9)血清抗体，HI滴度≥1:20的再用MN试验复核，MN滴度≥1:20的判为阳性。为考察可能的交叉反应，同时检测sH1N1、sH3N2以及LPAI H7N1抗体，结果按二分类变量形式纳入H7N9感染危险因素的单因素logistic回归检验。结果：（1）HI实验结果有27份标本（1.7%）HI抗体滴度≥1:20，再由MN复核，结果有8份标本（0.5%）MN滴度≥1:20。这8份抗-H7N9中和抗体阳性标本所对应的研究对象分别为无锡7个区（县）8个不同活禽市场的从业人员，其中7名活禽从业人员，1名为非活禽从业人员，H7N9抗体阳性率在活禽市场活禽从业人员中为2.3%（7/300），在非活禽从业人员中为0.1%（1/878）。这8名研究对象均否认一年内有流感样症状。（2）所有研究对象禽流感病毒H7N1的HI结果均为阴性。（3）二分类单因素logistic回归分析显示，活禽市场活禽从业人员感染H7N9的风险高于活禽市场非活禽从业人员（OR=20.95，95%CI2.57-171.01）。性别之间、不同年龄组之间、不同文化程度和不同收入水平之间抗体阳性率差异无统计学意义。sH1N1、sH3N2感染与H7N9感染也无统计学关联。结论：（1）Life Technologies VetMAXTM-Gold SIV Detection Kit和QuidelMolecular Influenza A+B assays对活禽肛拭子进行禽流感病毒H7N9检测的效果与WHO推荐的H7N9qRT-PCR assay基本一致。（2）江苏省无锡地区禽间AIVs感染情况复杂多样，有H5N1、H5N8、H9N2和H11N2等，H5N8和H11N2很可能是重组毒株。（3）职业暴露人群AIVs感染普遍，禽暴露人群中存在较高水平的禽流感病毒H5N1感染以及低水平的H7N9和H9N2感染，猪暴露人群中也存在低水平的H5N1和H9N2感染。这些感染者很可能是隐性或轻症感染。（4）工作场所为市场的禽类职业暴露人员、慢性呼吸道疾病以及年龄40-60岁为H5N1的感染危险因素。（5）活禽市场的活禽从业人员感染H7N9的危险高于活禽市场非活禽从业人员。因此，禽类职业暴露人群，特别是市场禽类从业人群，以及有呼吸道慢性疾病禽类暴露人群是AIVs的防控重点人群。加强活禽市场的管理、环境消毒和人员防护是控制AIVs传播的必要手段。对禽类和职业暴露人群AIVs感染状况进行系统、连续地监测有着重要的公共卫生意义。本研究的创新点包括：（1）用HI和MN两种方法串联，证实了活禽市场禽暴露人群中存在禽流感病毒H7N9隐性感染，而活禽市场以外禽暴露人群中没有发现H7N9隐性感染；（2）在同一禽暴露人群中发现多种AIVs隐性感染，包括H5N1、H7N9和H9N2感染；（3）发现禽暴露人群中禽流感病毒H5N1感染率较高；（4）发现猪暴露人群中存在低水平的禽流感病毒H5N1和H9N2感染；（5）发现活禽市场禽间AIVs感染复杂多样，并存在重组毒株。本研究的主要局限是血清学横断面调查无法确定研究对象AIVs感染发生的时间点，只能证明调查人群中可能存在既往感染。
[Abstract]:Human infection of avian influenza viruses (AIVs) has been reported more frequently in recent years. Since the avian influenza virus H5N1 was reported to be infected and killed in 1997, 658 people have been infected in 15 countries and the fatality rate is nearly 60%; the avian influenza virus H7N7 also caused 1 deaths in Holland in 2003; and other avian influenza viruses. The virus, such as H9N2, H7N3 and other infectious events in Hongkong, Canada and other places were reported in early.2013, and the case of avian influenza virus H7N9 infection appeared in the southeast of China. As of February 28, 2014, H7N9 had caused 375 people and 115 people died. Therefore, the AIVs infection among birds in China and the status of AIVs infection in the population are the scientific problems of concern.
In order to choose the fast and efficient detection method that can be applied to the field H7N9 avian influenza virus, we have 5 commercialized kits (Life Technologies VetMAXTM-Gold SIV DetectionKit, Quidel Molecular Influenza A+) to select the fast and efficient detection methods that can be used in the field of avian influenza virus in the field. B assay, Remel Xpect Flu AB assay, QuidelQuickvue influenza assay, and Quidel Sofia were evaluated first from the healthy live bird swab samples collected from the city of Wuxi and Nanjing, Jiangsu. QRT-PCR) influenza A assay and H7N9assay were tested for influenza A virus and avian influenza virus H7N9, and 27 positive specimens of H7N9, 70 H7N9 and influenza a negative specimens were screened. Then the 97 specimens were blinded by the above 5 kits and the WHO qRT-PCR H7N9assay was rechecked. Test principle, nucleic acid molecular detection method is repeated 3 times each, the method of antigen detection is repeated 2 times. Finally, WHO qRT-PCR H7N9assay is used as gold standard to calculate the sensitivity and specificity of these methods and their confidence interval (confidence interval, CI). Results: (1) the results of WHO qRT-PCR H7N9assay first and second rounds are in good agreement. (2) the results of Quidel Quickvue influenza assay were all negative for the test results of 97 specimens. The other four test methods had good specificity (96-100%). (3) Life Technologies VetMAXTM-GoldSIV Detection Kit specificity was the highest, 100% (95%CI0.87-1.00), false positive rate 4% (95%CI1-12%) sensitivity The rate was 85% (95%CI66-96%), and the false positive rate was 3% (95%CI0-10%).
In order to grasp the infection status and genetic evolution characteristics of AIVs in poultry in Wuxi area, we collected live fowl and its related environmental swabs in poultry and poultry farm of Wuxi city in July 2013 in -12 month, using RNA as a template extracted from swab specimens and using WHO qRT-PCR influenzaA assay to carry out the samples. The screening of influenza A virus was carried out by one step reverse transcriptional PCR for PCR amplification and gene cloning and sequencing of the influenza virus. Sequence analysis was carried out, and MDCK cells and chicken embryos were isolated and identified for the positive specimens of influenza A virus. Results: (1) collection of specimens. 305, the positive rate of influenza A virus was 28.5% (87/305). The positive rate of influenza A virus in poultry trading market samples was higher than that of farms (31.9%vs.10.4%, Chi 2=9.16, P=0.002); (2) there were 6 samples to be typed, respectively H9N2 (3), H5N8 (1), H11N2 (1) and H5N1 (1). Phylogenetic analysis showed that H5N8 and H11N2 had different degrees. Recombinant (3) a total of 3 strains of avian influenza virus were isolated: A/Chicken/1/Wuxi/2013 (H9N2), A/Chicken/2/Wuxi/2013 (H9N2) and A/Chicken/1/Wuxi/2013 (H5N1).
AIVs infection among birds is complex and diverse, while poultry raising, processing and other occupational exposures are high-risk groups of AIVs infection. In addition to infection of AIVs and even death, there are also latent infections such as H5, H7, H9, H11 and other subtype AIVs. Pigs can infect swine influenza virus (swine influenzavirus, SIV) and can infect a variety of birds. Source and human influenza viruses play the role of influenza virus intermediate host and multihost in the interspecific transmission chain of "avian - pig human". Studies have shown that pigs can infect avian influenza virus H5N1 and H9N2. Guangdong, Fujian, Zhejiang region, about 1% of porcine occupational exposure population have H9N2 infection. The infection status of influenza virus, through the analysis of the relationship between infection and the social and epidemiological characteristics of the general population, determines the risk factors of AIVs infection and provides the basis for the prevention and control strategy of human infection of AIVs. We have carried out influenza A influenza in the exposed population of poultry (pig) farms, slaughterhouses, markets and scattered households in Wuxi. A total of 498 avian occupational exposure, 608 porcine occupational exposures and 165 non occupational exposure controls were selected, and 1271 subjects were selected. The serum samples were tested by hemagglutination inhibition (HI) test and microneutralization (microneutralization, MN) test. H1N1 (seasonal influenza A/H1N1, sH1N1), sH3N2, and avian influenza virus H5N1, H9N2 and H7N1 serum antibodies were detected. Results: (1) occupational exposure in poultry was 17.7% (88/498), 10.9% (66/608) and 27.3%, respectively, and the positive rates of the three groups were different. The positive rates of.SH3N2HI antibody (chi 2=29.00, df=2, P=0.000) were 17.3% (86/498), 6.4% (39/608) and 8.5% (14/165), and there was a significant difference in the positive rate of sH3N2HI antibody in the three groups (x 2=34.29, df=2, P=0.000). (2) 14.6% and 8.4% of H5N1 in the occupational exposure population of poultry, respectively, in the occupational exposure of pigs. The group accounted for 0.2% and 0.2% respectively, and only 1 (0.6%) in the control group had H5N1MN titers of 1:80 and no higher titer. Multiple factor Logistic regression analysis showed that workplace exposed workers (OR=57.3,95%CI7.0-466.5), chronic respiratory disease (OR=12.8,95%CI1.6-102.5) and 40-60 years old (OR=3.1,95%CI1.5) -6.6) for the risk factors of H5N1MN's titer more than 1:80. (3) 76 sera of H5N1MN titer more than 1:80 were re done by HI test, and 3 H5N1 were determined according to the confirmed case standard of WHO MN titer > 1:80 and HI titer > 1:160. It was 2 market poultry personnel and 1 large-scale poultry farm workers, accounting for 0.6% of the poultry exposed population, and 2 of these 3 people. Influenza like symptoms were denied in the last year. (4) the H9N2 test results showed that 5 subjects had the MN titer more than 1:10, of which 2 were market selling poultry, 2 were market selling pork personnel and 1 were pigs slaughterhouse workers.H9N2 in poultry occupational exposure and pigs exposed to MN antibody positive rates of 0.4% (2/498) and 0.5% (3/608) respectively. The control population was not in the control population. H9N2MN antibody positive was found. (5) H7N1HI test results showed no antibody titer was greater than 1:10.
In early 2013, the H7N9 avian influenza virus had been infected with human cases in southeastern China and was growing rapidly in the short term. Exposure to live birds and related environments, including live poultry market, was considered as the primary risk factor for H7N9 infection. In the duration of the H7N9 epidemic, is there a recessive infection in the exposed population of birds and the living fowl practitioners and non people in the live poultry market Is there the same risk of infection among live poultry workers and poultry workers outside the live poultry market? What are the relationships between infection and social demography, basic disease, occupational behavior and other factors? Based on these scientific problems, we conducted a serological survey of H7N9 in the Wuxi avian occupational exposure population. 1588 participants, including 1178 live poultry market practitioners (300 live bird practitioners and 878 non live poultry employees), 245 live poultry workers outside the live poultry market and 165 local health residents, selected Turkey blood HI to detect A/Anhui/1/2013 (H7N9) blood serum antibody, HI titer more than 1:20, MN test recheck, MN titer The sH1N1, sH3N2, and LPAI H7N1 antibodies were examined for the possible cross reaction, and the results of the single factor Logistic regression test of the risk factors of H7N9 infection were examined in the form of two classified variables. Results: (1) the results of the HI test had 27 specimens (1.7%) of HI anti body titer more than 1:20, then rechecked by MN, and 8 specimens (0.5%) M. The research objects of the 8 anti -H7N9 neutralization antibody positive specimens of N titer more than 1:20. were employed in 8 different live poultry markets in 7 districts and counties in Wuxi, of which 7 live poultry workers and 1 non live poultry workers, and the positive rate of H7N9 antibody was 2.3% (7/300) among live poultry workers in live poultry market and in non living poultry workers. 0.1% (1/878). All 8 subjects denied influenza like symptoms in one year. (2) all the HI results of avian influenza virus H7N1 were negative. (3) two classified single factor Logistic regression analysis showed that the risk of living poultry workers infected with H7N9 was higher than that of non live poultry workers in live poultry market (OR=20.95,95%CI2.57-171.01). There was no significant difference in the positive rate of antibody positive rate between different age groups, different age groups, different educational levels and different income levels, but there was no statistically significant correlation between sH3N2 infection and H7N9 infection.
Conclusion: (1) the effect of Life Technologies VetMAXTM-Gold SIV Detection Kit and QuidelMolecular Influenza A+B assays on the H7N9 detection of avian influenza virus in live bird and anal swabs is basically the same as that recommended by WHO. 11N2 is likely to be a recombinant strain. (3) AIVs infection in occupational exposure population is common, high levels of avian influenza virus H5N1 infection and low level of H7N9 and H9N2 infection are present in the exposed population of birds, and low levels of H5N1 and H9N2 infection are also present in the exposed population of pigs. These infected people can be recessive or mild infection. (4) the workplace is the market. Occupational exposures for poultry, chronic respiratory diseases and 40-60 years of age of 40-60 years as risk factors for infection. (5) the risk of living poultry workers in the live poultry market is higher than that of non living poultry workers in the live poultry market. Therefore, occupational exposure to poultry workers, especially in the market birds, and of chronic respiratory diseases in the respiratory tract. The exposed population is the key group of prevention and control of AIVs. Strengthening the management of the live poultry market, environmental disinfection and personnel protection are the necessary means to control the transmission of AIVs. The system of AIVs infection in poultry and occupational exposed population has important public health significance.
The innovative points of this study include: (1) in series with two methods of HI and MN, it confirmed the existence of H7N9 recessive infection of avian influenza virus in the poultry exposed population of live poultry market, and no H7N9 recessive infection was found in the poultry exposed population outside the live poultry market; (2) multiple AIVs infection in the same bird exposed population, including H5N1, H7N9 and H9N2 infection, was found. (3) the H5N1 infection rate of avian influenza virus was found in the exposed population of birds. (4) there was a low level of avian influenza virus H5N1 and H9N2 infection in the exposed population of pigs, and (5) it was found that the AIVs infection among birds in the live poultry market was complex and varied, and there was a recombinant strain.
The main limitation of this study is that the serological cross-sectional survey can not determine the time point of the AIVs infection, and it can only prove that there may be previous infection in the survey population.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R511.7

【参考文献】