Case-control study of risk factors for human infection with influenza A ( H 7 N 9 ) virus in Jiangsu Province , China , 2013

J Ai1, Y Huang2,3, K Xu1, D Ren3,4, X Qi1, H Ji1, A Ge1, Q Dai1, J Li1, C Bao1, F Tang1, G Shi3, T Shen3, Y Zhu (jszyf@jscdc.cn)1, M Zhou1, H Wang1 1. Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, Jiangsu, China 2. Tongling Center for Disease Control and Prevention, Tongling, Anhui, China 3. Chinese Field Epidemiology Training Program, Chinese Center for Disease Control and Prevention, Beijing, China 4. Tongren Center for Disease Control and Prevention, Tongren, Guizhou, China

Since it was discovered in February 2013, the emerging outbreak of human infections with a novel avian influenza A(H7N9) virus in China, has raised serious concerns for public health throughout the world [1].According to reports by China National Health and Family Planning Commission, a total of 130 confirmed cases of human infection including 37 deaths (28%) were reported in mainland China as of 27 May 2013.Few descriptive studies have been reported on risk factors associated with avian influenza A(H7N9) virus infection [1,2].We conducted a matched case-control study to identify potential risk factors for human influenza A(H7N9) cases in Jiangsu Province, one of the areas where the novel reassortant influenza A(H7N9) virus emerged earliest.

Selection of cases and controls
All confirmed human cases of avian influenza A(H7N9) virus infection in Jiangsu Province were included in this study.Cases were defined according to The diagnosis and treatment programs of human infections with H7N9 virus issued by the National Health and Family Planning Commission of the People's Republic of China [3].As of 27 May 2013, 27 confirmed cases of human infection with influenza A(H7N9) were reported in Jiangsu; of these, eight cases were fatal.
We matched four neighbours as controls for each patient.The selection criteria included: testing negative for influenza A(H7N9) virus, same sex, no more than five-year age difference, and no respiratory tract symptom like cough or sore throat, or other symptoms like fever (≥38 o C) within two weeks of illness onset of the matched case.Cases were classified according to the area in which they lived: rural or urban.For cases from rural areas in the Province, controls were recruited from each patient's village according to the principle of proximity.The nearest neighbours were recruited first; if there were no eligible controls, additional controls were recruited among more distant neighbours.For cases living in urban areas, the apartment building where the patient lived was selected first.The persons in the same unit of the building were recruited first; if there were no eligible controls, the neighbours of the nearest unit were recruited next, and additional controls were recruited from the closest apartment building if needed.Investigators recruited the potential controls by visiting their homes.After the recruitment, all the eligible controls were asked to visit the local clinics for interview and blood sample collection.From each control 5 ml venous blood was collected to exclude unapparent and/or past influenza A(H7N9) virus infection.The study was approved by the Ethics Committee of the Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu CDC).Before the investigation, we obtained the informed consent of all the participants in the interview.

Data collection
We performed an epidemiological survey for all participants including demographic characteristics, health status, daily habits, and other potential risk behaviour, such as not washing hands frequently (i.e.before meals and after using toilet), smoking, etc. Interview questions were also related to the activities and environment-related exposures for both cases and controls within two weeks before the illness onset of the case.Environment-related exposure was defined as possible exposure due to the environment, including visiting live poultry market, bird market, buying freshly slaughtered poultry or birds in live poultry market, and chickens or pigeons raised in the neighbourhood.Direct contact included direct contact with live poultry or birds in the wet market (i.e. through culling and slaughtering), direct contact at home (i.e. through cleaning, processing or cutting), and occupational exposure.For fatal cases of influenza A(H7N9) infection, or for the patients too ill to participate in the interview, a family member (usually the spouse) responded to the questions for the purpose of this study.The interview was carried out by trained staff of the Jiangsu CDC and trainees of the Chinese Field Epidemiology Training Program.

Laboratory investigation
Serum samples from controls were tested by haemagglutination inhibition assay with turkey red blood cells against avian influenza A(H7N9) virus strain (A/ Jiangsu/XWQ/2013), with two confirmed patients' series serum as positive reference.All tests were performed in Jiangsu CDC.

Statistical analyses
Data were analysed using Stata/SE 9.0 for Windows (Stata Corp LP, College Station, TX, USA).Each study factor was compared between cases and controls using exact conditional logistic regression.Further multivariate conditional logistic regression was performed including all variables with P≤0.10 in univariate analyses and backward method was applied to exclude the variables with P>0.10.

Case-control study results
A total of 25 confirmed human influenza A(H7N9) cases were included in the study.Two cases were excluded: one was possibly infected by human-to-human transmission (data not shown), and for the second one data were insufficient.All 25 cases included were positive in real-time PCR for influenza A virus subtype H7N9, and 16 cases were positive in virus isolation.
The epidemiological survey was conducted from 25 April to 12 May 2013.Ninety-three controls were enrolled in this study: 22 cases had four controls matched for each, and the remaining three cases only had one, two, and two controls matched, respectively, due to unavailability of eligible controls according to the control selection criteria.All enrolled controls were seronegative for avian influenza A(H7N9) virus antibodies.Data for 13 patients (including the eight fatal cases and the five severe cases) were obtained from family members, while data for the remaining cases and all controls were provided by themselves.
The demographic characteristics of cases and controls are showed in Table 1.The age of the patients varied between 21 and 85 years with a median of 56 years.Twelve patients were older than 60 years and 19 were male.Twenty-two cases lived in the urban area.The characteristics of cases and controls were similar in terms of education level, family size, and family income per capita.Two patients worked in a wet poultry market (slaughtering and selling poultry) and one patient and one control worked in poultry transportation.Fifteen patients and 39 controls had chronic medical diseases, including hypertension, diabetes, cancer, chronic bronchitis, rheumatic arthritis, and coronary disease (Table 1).
In the univariate analysis, the following exposures were associated with a high risk of infection with influenza A(H7N9): direct contact with poultry or birds within two weeks before illness onset (direct contact with poultry or birds in live poultry market, direct contact through preparing and cooking poultry or birds at home); environment-related exposures (visiting the market where live poultry was commercialised, buying freshly slaughtered poultry or birds in live poultry market).Frequent hand washing was associated with a reduced risk of getting infected with influenza A(H7N9) virus and this habit was reported more often by controls (89%) than by cases.Body Mass Index (BMI) (≥25), having ever smoked, and travel history were not significantly different between cases and controls (Table 2).
Although a higher proportion of chronic medical diseases was noticed in cases than in controls, the difference was not statistically significant (P>0.05).
Coronary disease 1 NA: not available.
a Comparison of frequencies between cases and controls was analysed by exact conditional logistic regression; matched factors (age, sex, and location) were excluded from analyses.b For the fatal cases or for those too ill to respond to the survey, a family member (usually the spouse) responded to the survey.c Information not available because of small sample size or because data distribution was not suitable for conditional logistic regression model.
Hypertension was high in both groups, the proportion was 40% among cases and 33% among controls (P>0.05).By excluding hypertension, 48% of cases had chronic medical diseases compared to 20% of controls, resulting an OR of 5.1 (95% CI 1.5-16.9;P =0.008).
In the multivariate conditional logistic regression model, three variables represented significant risk factors for human influenza A(H7N9) virus infection: direct contact with poultry or birds, chronic medical conditions (hypertension excluded) and environment-related exposures (Table 3).
species of live poultry and birds in live poultry markets in China creates a dynamic micro-environment that favours interspecies transmission [9].In wet poultry markets, the secretions, faeces and organs of poultry were splashed out, which made the surroundings easily contaminated and more likely be exposed to infected poultry.Surveillance of live poultry markets in epidemic regions indicated that the poultry cages were positive for avian influenza A(H7N9) virus [10].
The univariate analysis showed that for persons who went to live poultry markets more frequently (>10 times within two weeks of illness onset) the risk of exposure to influenza A(H7N9) virus was significantly higher and this could be due to the frequency of exposures.In addition, inhalation of aerosolised material contaminated with influenza A(H7N9) viruses may be a possible transmission route; however, our study did not investigate this potential transmission vehicle.
Different from influenza A(H5N1) virus infection, our findings show that having an underlying medical condition/co-morbidities (hypertension excluded) was significantly associated with influenza A(H7N9) virus infection.Age increases the risk for chronic diseases, which has been well-matched between cases and controls in this study.Therefore, a high proportion of elderly patients with severe influenza A(H7N9) virus infection may be due to decreased immune function caused by underlying chronic diseases [9].
Although influenza A(H7N9) virus is highly pathogenic in humans, poultry infected with it do not develop any obvious or visible diseases.It is thus difficult to separate the infected poultry or birds from the un-infected ones.People, especially those with underlying diseases, such as chronic bronchitis, diabetes, cancers, rheumatic arthritis, and immunodeficiency disease, could avoid the risk by reducing exposure to possibly contaminated environment.Persons exposed to poultry are advised to undertake appropriate protection and to develop correct hand washing habits.
Our study has several potential limitations.First, the number of cases was small, limiting the power of the study to demonstrate significant associations.Second, for some cases, family members responded to the interview and it is possible that they were less likely to be aware of specific exposures and activities, and this may result in bias to the data.Third, recall bias may be included as well since participants in the study were asked about activities that had occurred one month before the survey.Fourth, as residence was one of matching factors for cases and controls in this study, this could lead to some underestimation of the risk as the neighbourhood may share some living environmental exposures with cases.
In conclusion, ongoing education of the public is needed for behavioural changes in order to decrease the exposure risk to influenza A(H7N9) virus.Additionally, effective control strategies should be implemented; the most drastic measure would be closing the live poultry markets but if this is not possible, other measures such as segregating bird species, improving biosecurity, and having centralised poultry slaughtering locations, regular disinfection, and a periodical rest day, should be considered [11,12].In addition, wearing protective masks or respirators by workers and visitors to wet poultry markets could be discussed.

Table 1
Baseline characteristics of participants in a case-control study of avian influenza A(H7N9) in Jiangsu Province, China, 2013