The influence of urban livestock in Hanoi, Viet Nam, on dengue epidemiology

Urbanization and livestock intensification are closely connected. Livestock are brought to the cities during urban migration and provide an important source of food and income for the farmers. With more humans and animals living close together, vectors such as mosquitoes may increase as well as cause an increased transmission of infectious diseases such as dengue. This study investigates how the presence of mosquito vectors are affected by the keeping of livestock in urban environments and evaluates the dengue awareness in the city of Hanoi, Viet Nam. In two districts, Ha Dong and Dan Phuong, 140 households with and without livestock were interviewed followed by the collection of mosquitoes and larvae. A panflavivirus qPCR was used to test pools of mosquitoes for the presence of flavivirus. A general trend was observed from the KAP study; respondents living in Dan Phuong district had better knowledge and practice regarding dengue compared to Ha Dong. In total, 3899 mosquitoes were collected, whereupon 52 (1.33%) of them were of Aedes species. A significant district difference was observed, with households living in Ha Dong having more frequent presence of Aedes spp. mosquitoes (p=0.02) and higher reported incidence of dengue (p=0.001). There were no significant association between livestock keeping and the presence of Aedes spp. mosquitoes (p=0.955) or between livestock keeping and the incidence of dengue (p=0.08). Nevertheless, it is still of great importance to continue investigating the association between mosquitoborne infections and urban livestock keeping and the possible challenges in the future

The influence of urban livestock in Hanoi, Viet Nam, on dengue epidemiology Frida Jakobsen Master Degree Project in infection biology, 30 credits Spring 2018 Department: International Livestock Research Institute (ILRI)/IMBIM Supervisor: Johanna Lindahl Co-supervisor: Hung Nguyen and Thang Tien Nguyen UPPSALA UNIVERSITET (38) Table of content Abstract Mosquitoes, livestock and dengue fever – will urbanization be the demise of mankind? Introduction Dengue Aedes aegypti & Aedes albopictus Hanoi, Viet Nam Aims Materials and Methods Ethical considerations Study design Study site Questionnaire and interviewing 11 Mosquito and larvae collection 12 Mosquito and larvae identification 12 RNA extraction 12 Pan-flavivirus qPCR & gel electrophoresis 12 Statistical methods 13 Results 14 Dengue awareness study 14 General information of the households 14 Livestock information 15 Dengue and mosquito knowledge 17 Dengue attitude 19 Dengue and mosquito practice 20 Mosquito and larvae collection 22 Dengue fever cases 22 Pan flavivirus q-PCR and gel electrophoresis 24 Discussion 25 Acknowledgements 28 Appendices 33 1.The questionnaire 33 2.Informed consent form 38 UPPSALA UNIVERSITET (38) Abstract Urbanization and livestock intensification are closely connected Livestock are brought to the cities during urban migration and provide an important source of food and income for the farmers With more humans and animals living close together, vectors such as mosquitoes may increase as well as cause an increased transmission of infectious diseases such as dengue This study investigates how the presence of mosquito vectors are affected by the keeping of livestock in urban environments and evaluates the dengue awareness in the city of Hanoi, Viet Nam In two districts, Ha Dong and Dan Phuong, 140 households with and without livestock were interviewed followed by the collection of mosquitoes and larvae A pan-flavivirus qPCR was used to test pools of mosquitoes for the presence of flavivirus A general trend was observed from the KAP study; respondents living in Dan Phuong district had better knowledge and practice regarding dengue compared to Ha Dong In total, 3899 mosquitoes were collected, whereupon 52 (1.33%) of them were of Aedes species A significant district difference was observed, with households living in Ha Dong having more frequent presence of Aedes spp mosquitoes (p=0.02) and higher reported incidence of dengue (p=0.001) There were no significant association between livestock keeping and the presence of Aedes spp mosquitoes (p=0.955) or between livestock keeping and the incidence of dengue (p=0.08) Nevertheless, it is still of great importance to continue investigating the association between mosquito-borne infections and urban livestock keeping and the possible challenges in the future Key words; Knowledge, attitude, practice, Aedes mosquitoes, Ha Dong, Dan Phuong, Panflavivirus qPCR UPPSALA UNIVERSITET (38) Mosquitoes, livestock and dengue fever – will urbanization be the demise of mankind? The popular summary of the project “the influence of urban livestock in Hanoi, Viet Nam, on dengue epidemiology” People living closer to the city of Hanoi are more likely to get infected with dengue fever – one of the most important mosquito-borne diseases present worldwide that is affecting many hundred million people each year Moving closer to the cities is an increasing trend among people all over the globe and their animals will follow in the owner’s footstep Nevertheless, with more humans and animals living in a limited area, the numbers of vectors such as mosquitoes may increase, causing infectious diseases such as dengue fever to spread more rapidly Garbage and man-made containers are expected to be found in the gardens and around the households when keeping livestock, thus creating perfect homes for mosquito larvae to thrive in This study aims to explore the interactions between the disease-carrying mosquitoes, and livestock keeping in urban environments Another important aim of this study is to investigate the knowledge, attitude and practices among residents living in two districts in Hanoi city, Viet Nam An urban district, Ha Dong, with a lower livestock population density, and a peri-urban district, Dan Phuong, with a higher livestock population density, was selected to participate in this study In total, 140 households with and without livestock were interviewed about their awareness of dengue fever The households were also searched for mosquitoes and mosquito larvae both inside and outside the households The mosquitoes were later investigated in order to see if they contained any dengue virus The results from this study show that livestock keeping in urban environments are not an important factor for being infected with dengue fever Instead, where you live seems to be a more important aspect, since 15 out of 17 households that had been infected with dengue virus were located in the urban district Ha Dong Also, more dengue transmitting mosquitoes were found in their households On the other hand, the people living in the area with less dengue fever had better knowledge, attitude and practices regarding dengue fever compared to the people living in Ha Dong Furthermore, no dengue virus was found in the mosquitoes that were collected, which can most reasonably be explained by the fact that we collected the mosquitoes during the dry and cool period, when outbreaks are not occurring Our results indicate a need for further research on vector-borne diseases targeting risk factors associated with urbanization and the increasing livestock population density in the cities The fictive question if urbanization will be the demise of mankind, is perhaps not so far-fetched after all, only future research can tell UPPSALA UNIVERSITET (38) Introduction Infections pose a huge threat to humans worldwide While diseases may emerge anywhere in the world, infectious diseases are more prevalent in tropical areas such as Southeast Asia, which usually is referred to as a hotspot for emerging and re-emerging infectious diseases (Coker et al., 2011) About 23% of the emerging infectious diseases are vector- and arthropodborne, and mosquitoes are one of the medically most important vectors There are several arthropod-borne infections, including zika, Japanese encephalitis (JE) and dengue, which are all causing human suffering The exposure frequency of mosquitoes, humans and animals have increased due to a fast-growing population, urbanization, globalization, livestock intensification, more international trade and travelling, deforestation, and a changing climate (Gould et al., 2017; Lindahl & Grace, 2015; Coker et al., 2011) More than half of the population worldwide live in urban settings (The World Bank, 2016) Livestock is brought to the cities during urbanization and is an important source of food in the cities and serve as an income for many farmers However, the livestock may also function as reservoirs for many infections and might as well increase the presence of mosquito vectors The keeping of livestock may create more breeding sites for mosquitoes due to incomplete sanitation, increased water usage and provide ample opportunities for blood feeding Dengue Dengue, or dengue fever, is an arthropod-borne viral disease belonging to the genus of Flavivirus and is transmitted between humans by Aedes mosquitoes (WHO, 2012) There are two diverse transmission cycles that maintains dengue virus (DENV) endemic; a human cycle and a sylvatic cycle The human cycle includes the transmission between Aedes mosquitoes and humans, where humans acts as the only known reservoirs The sylvatic cycle involves nonhuman primates and Aedes mosquitoes (Vasilakis et al., 2011) Dengue was ranked the most important mosquito-borne viral disease in the world in 2012 and outbreaks occur throughout most tropical countries (WHO, 2012) DENV is a positive sense single-stranded RNA virus with four different serotypes; DENV 1-4 The serotypes are spread globally and several genotypes have been recognised within each serotype The disease can manifest in a wide range of clinical symptoms and the severity of dengue vary from asymptomatic to severe dengue The typical symptoms of dengue include fever, headache, nausea, rashes and muscle-pain (WHO, 2009; WHO, 2012) Even though the majority of cases are non-severe, some cases develop into dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) (Bhatt et al., 2013) Severe dengue is characterized by at least one of the three following: (I) plasma leakage or accumulation of fluids, with/without respiratory distress, or (II) severe bleeding or (III) severe impairment of the organs (WHO, 2009) The diagnosis of dengue can be challenging since the symptoms resemble several other febrile illnesses and the diagnosis can therefore not solely rely on clinical symptoms Laboratory diagnosis to confirm the disease is of importance for the management of the patient, as well as for surveillance and outbreak control Methods for laboratory confirmation may include molecular methods for viral detection (virus or nucleic acid) and serological methods for detection of antigens or antibodies (WHO, 2009) Most patients not require hospital care, but it is important to recognize the disease early to reduce dengue mortality The majority of the deaths from dengue is a result of severe shock The management of dengue vary depending on the severity, but the treatment is symptomatic in combination with close monitoring of the patient to be able to observe any warning signs After the primary infection, the patient gets lifelong protective immunity of the UPPSALA UNIVERSITET (38) infecting serotype, nevertheless, the patient can still be infected by the other dengue serotypes (WHO, 2009) An estimated 3.97 billion people are at risk of being infected with dengue in 128 countries around the world, which creates a powerful demand for effective vaccine development (Brady et al., 2012) The first dengue vaccine, CYD-TDV (Dengvaxia®), was licensed in 2015 Dengvaxia® is a recombinant, live-attenuated, tetravalent vaccine developed by Sanofi-Pasteur and has been approved for children above years of age in several endemic countries in Asia and Latin America (Vigne et al., 2017; WHO, 2017) However, there is a concern that vaccinated seronegative individuals experience a higher risk of getting severe dengue through antibody dependent enhancement (ADE) via the sensitization by the vaccine (Halstead, 2017) ADE occurs when antibodies from a primary infection or in this case from the vaccination, binds to subsequently infecting dengue virus of another serotype The primary antibodies will not be able to neutralize the virus and instead Fc-γ receptor-bearing cells will be bound by the antibody-virus complex Thereby, the antibodies will help the viral replication, since the dengue virus would otherwise inefficiently infect these cells ADE therefore increases the risk of developing severe dengue and a great concern when developing a successful vaccine (Whitehead et al., 2007) About 200 years ago, there was a low prevalence of dengue virus The prevalence did not increase until there was a huge expansion of the population globally, which generated enough susceptible hosts, together with more international travelling and spread of vectors, the virus was able to sustain in the human population (Zanotto et al., 1996) The incidence of dengue has increased 30-fold over the past 50 years and the global burden of dengue is today predicted to be 390 million (284-528, 95%) infections each year Of these, 96 million (67-136) are apparent infections of different levels of severity and Asia suffer about 70% of the infections (Bhatt et al., 2013; WHO, 2012) Even though Asian countries are most affected by dengue, many Europeans experience dengue infections after travelling abroad In fact, dengue is the second most common reason in Europe for hospital admission after travel, with 1143 confirmed cases in 2010 A large outbreak of DENV1 was also reported in the Portuguese autonomous region of Madeira between October 2012 and January 2013 After infected people arrived at the isle, the combination of mosquito vectors and a high population density of naïve individuals resulted in more than 2200 confirmed cases (Schaffner & Mathis, 2014) Dengue was first reported in Viet Nam in 1959 and dengue has since then become endemic across the country (WHO, 2010) A national program for Dengue Surveillance and Control exists, which includes all provinces of Viet Nam since 2005 (Vu et al., 2014) Viet Nam had among the highest cumulative incidence of dengue (144,69 incidence/100 000 population) in the western pacific region in 2010 Other countries with a high cumulative incidence included the Philippines, Singapore, Malaysia and Laos (WHO, 2010) Even though dengue is endemic in Viet Nam, the incidence differs across the country The southern parts of the country have a higher number of cases compared to the north Actually, the mean incidence rates were 3-7 times lower in Hanoi compared to Khanh Hoa, Ho Chi Minh and An Giang (all located in the south) There is also a seasonal pattern of dengue in Viet Nam, with the highest incidence rates in May to December, during the rainy season (WHO, 2018; Lee et al., 2017) Several outbreaks of dengue have been reported in Viet Nam over the years and major outbreaks have occurred around every ten years (1987, 1998, 2009) The 2009 outbreak in Hanoi reported 16 263 dengue cases, with 121 cases per 100 000 people, which was a 6.7 times increase compared to the same time in 2008 The latest dengue outbreak in Hanoi occurred in 2017 with 37 651 number of cases and deaths (Hanoi preventive medicine centre, 2017; Minh An & Rocklöv, 2014) UPPSALA UNIVERSITET (38) Aedes aegypti & Aedes albopictus The virus is transmitted by Aedes mosquitoes, primarily Aedes aegypti and Aedes albopictus After the female mosquito ingest the virus during a blood meal, the virus spreads systemically within the mosquito during 8-12 days Amplification of the virus occurs in the salivary glands and the mosquito can subsequently transmit the virus to new hosts during feeding (WHO, 2009; Raquin & Lambrechts, 2017) Aedes aegypti is considered to be the main vector in urban areas, where the mosquitoes mainly breed in man-made water storage containers, e.g water tanks, jars, bonsai trees and old car tires (Huber et al., 2003) Aedes albopictus has more recently become abundant in urban and peri-urban areas Further geographical expansion of Aedes albopictus is expected in the near future, mainly due to urbanization, transportation of larvae/eggs in containers, and a changing climate (Faraji & Unlu, 2016) The most efficient way to reduce the burden of dengue infection has been to control the mosquito vectors, since no vaccine has previously been available Vector control is mainly performed through integrated vector management programmes using insecticides, environmental management and through increasing the public awareness about dengue (Bilal et al., 2017) A global strategy for dengue prevention and control has also been defined by the World Health Organization (WHO) where the goal is to reduce the burden of dengue by 2020 to combat the growing problems with dengue worldwide (WHO, 2012) There have been discussions regarding the effectiveness of the Aedes mosquito control programs The general perception that the control programs has not reduced dengue transmission have been questioned by Bowman et al (2016) They argue that this perception is incorrect and that the real problem is the lack of reliable evidence needed to make these conclusions about the existing control programmes (Bowman et al., 2016) Nevertheless, dengue is still re-emerging and there is a growing concern regarding increasing resistance against insecticides for both Aedes aegypti and Aedes albopictus Insecticide resistance has been reported in several countries in Asia, e.g India, China and Thailand (Chatterjee et al., 2018; Yiguan et al., 2016; Jirakanjanakit et al., 2007) Therefore, dengue remains a public health issue worldwide that is in need of efficient control Hanoi, Viet Nam Viet Nam is a tropical country in the western Pacific region In 2016, 34% of the total population were living in urban settings in Viet Nam, a number that keep increasing each year (The World Bank, 2016) The capital, Hanoi, is the second most populous city with an average population of 7.3 million people (GSO, 2016) There is also a high livestock population in Hanoi with 24.8 thousand buffaloes, 136.8 thousand cattle (beef and dairy), 1.9 million pigs and 23.6 million poultry according to the latest livestock population data from Hanoi subdepartment of animal health Several studies have reported weather and climate components as important risk factors for dengue incidence and vector survival in Viet Nam (Xuan et al., 2014; Pham et al., 2011; Do et al., 2014; Vu et al., 2014) Depending of the geographical location, southern or northern parts of the country, the different weather components were either positively or negatively correlated to dengue incidence One example is the positive association between dengue incidence and rainfall in Hanoi While a negative association was found in Ba Ria Vung Tau (Vu et al., 2014) Other findings in Viet Nam suggest that keeping an animal shelter, a garden and garbage close to the households were associated with a high incidence of dengue Also, farmers had an almost times higher risk of getting infected with dengue (RR 7.94; 95% CI 2.29-27.55) (Lan Phuong et al., 2008) It has also been suggested that there is an association between the presence of UPPSALA UNIVERSITET (38) dengue IgG antibodies and the management of pigs The reason for this association may be due to the formation of habitats for Aedes larvae when raising pigs However, there is a huge knowledge gap in this area and there is a strong need for further investigation (Thai et al., 2005) Although dengue virus is mainly transmitted between humans through mosquitoes, high human population density, together with a high livestock population density may increase the transmission of several infectious diseases, including dengue, for example through the formation of larval habitats when keeping livestock As previously mentioned, the best method for controlling dengue is to control the mosquito vectors Hence, it is of great importance to get a better understanding about the interactions between dengue mosquito vectors and the keeping of the livestock in urban settings Aims The high number of livestock and humans in close contact may increase the risk of dengue transmission, particularly if the livestock keeping is associated with the presence of larval habitats and increasing number of mosquitoes It is therefore of importance to understand how the presence of dengue vectors are affected by the keeping of livestock in urban areas since previous studies are scarce The aim of this study is to investigate how the presence of dengue vectors (Aedes aegypti and Aedes albopictus) is affected by the keeping of livestock in urban environments in Hanoi, Viet Nam The aim was also to get a better understanding of the awareness of dengue in the city of Hanoi This was investigated through interviewing people about their knowledge of dengue fever and the housing of their livestock Subsequently, inspection of local households for mosquitoes and larvae followed by detection of dengue virus by a qPCR detecting flavivirus was performed UPPSALA UNIVERSITET (38) Materials and Methods Ethical considerations The study protocol was approved by the ethical committee of Hanoi University of Public Health (HUPH) (approval reference No.: 048/2018/YTCC-HD3) All participants were informed that the participation in the study was voluntary and that they have the possibility to drop out of the study at any time A written informed consent was obtained from all of the participants before conducting the interview Study design To get a better understanding of the dengue awareness of households in urban and periurban settings of Hanoi, Viet Nam, a cross-sectional study on knowledge, attitudes and practice (KAP) was performed in two districts of Hanoi Study site Two districts were selected for sample collection based on livestock population data from 2017 from Hanoi sub-department of animal health Ha Dong is located in the central parts of Hanoi (urban area) with low livestock population density, and Dan Phuong is a peri-urban area that has a higher livestock population density (see figure and 2) Figure The population density of livestock and humans in the districts of Hanoi city (population/km2) The data are obtained from 2017 from Hanoi sub-department of Animal health A Cattle population density in Hanoi B Poultry population density in Hanoi UPPSALA UNIVERSITET 10 (38) A B Figure The population density of livestock and humans in the districts of Hanoi city (population/km2) The data are obtained from 2017 from Hanoi sub-department of Animal health A Pig population density in Hanoi B Human population density in Hanoi Sample size and selection of households To calculate the sample size, a 10% prevalence of dengue vectors in the households were presumed With an anticipated precision of and a 95% confidence level, the sample size was decided to be 139 households This sample size would also be enough to detect a difference of 20% with a power of 80% between the non-livestock and the livestock keeping household In the two districts, 70 households were interviewed and the same number of livestock keeping and non-livestock keeping households were identified through selection of random GPS points and from the help of local commune veterinarians 16 random GPS points were identified in each district using ArcGIS 10.4 and the distance between each point was set to 1.5 km Out of these 16 random GPS points, 12 were randomly selected in each district to generate 72 households per district (see figure 3) In Ha Dong, the 12 selected points were located in central areas where there were no livestock present, hence, all 12 selected points were not used Instead, local veterinarians identified communes and households with livestock Therefore, only GPS points (7-12 households per point) were included to generate a total number of 70 households In Dan Phuong, close to the GPS points, the veterinarians randomly selected households with and without livestock for participation in the study This approach was possible since Dan Phuong has a higher livestock population density Three households without and three households with livestock were identified for each GPS point For both district, if the selected household was not willing to participate, the interviewer continued until a consenting household was found UPPSALA UNIVERSITET 24 (38) Number of households 14 12 10 Figure 10 The number of households that had been infected with dengue fever for different time periods of the year Pan flavivirus q-PCR and gel electrophoresis In total 193 samples were extracted The pan flavivirus q-PCR of the extracted RNA with suspected positive CT-values and melting temperatures were further investigated through gel electrophoresis 170 samples were run in the qPCR and out of these, 65 samples (38.24%) of the samples had suspected positive results and were therefore run in the gel electrophoresis However, none of the samples showed a band in the gel electrophoresis except for the positive controls, hence no positive samples were found (see figure 11) Figure 11 One of the gel electrophoresis picture showing all negative samples except for the positive control, that is showing a clear band UPPSALA UNIVERSITET 25 (38) Discussion To get a better understanding regarding the dengue fever awareness in Hanoi, Viet Nam, 140 households in two districts, Dan Phuong and Ha Dong, were interviewed using a structured questionnaire A general trend was observed; people living in Dan Phuong had better dengue knowledge, attitude and practice compared to the people living in Ha Dong It was also observed that health care worker (p=0.029) and loudspeaker (p