Asssessing the potential of Arboviral Transmission in Karonga District, Northern Malawi

Asssessing the potential of Arboviral Transmission in Karonga District, Northern Malawi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Asssessing the potential of Arboviral Transmission in Karonga District, Northern Malawi Penjani Promise Redson Chunda This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6883143/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This entomological study carried out in Karonga aimed at establishing arbovirus vector densities in communities and arbovirus presence in the vectors. Adult mosquitoes were trapped using oviposition traps (ovitraps), and aquatic stages were sampled using nets and/ or scoops and were subsequently reared to the adult stage. Aedes aegypti was prevalent in all the six study sites. However, the study did not find Aedes albopictus, which is another known potential vector. The average Aedes Aegypti densities per site were: Hara 82 (15.7%), Iponga 144 (27.6%), Kaporo, 165 (31.6%), Karonga Town 128 (24.5%), and Kayelekera 3 (0.6%). There was an increase in density going further north of Karonga which borders Tanzania. Polymerase Chain Reaction (PCR) showed that all sites had dengue (DENV) and Chikungunya (CHIKV) viruses, implying that in sampling sites, communities are at direct risk of dengue, Chikungunya infection, and other arboviruses. Further studies are required to fully understand and characterize the extent of arboviruses among local communities and the role of Aedes aegypti in their transmission. There is an urgent need to set up the laboratory platforms, monitoring, surveillance and control systems for nation by the Public Health Institute of Malawi (PHIM) which is responsible for disease surveillance and response before arboviral diseases such as Dengue, Zika, Chikungunya, and others become epidemic and a major public health problem in the country. Virology Entomology Arboviruses Vector Dengue Chikungunya Aedes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 CHAPTER ONE BACKGROUND 1.1 Background Information Historically, Chikungunya Virus (CHIKV) was first isolated in a febrile case in the southern part of Tanzania during a dengue-like outbreak between 1952 and 1953 (WHO et al., 2016). Chikungunya Viruses belong to a genus called Old World Alpha viruses and family Togaviridae. It is an Icosahedral virus encased by lipids and is a single-stranded RNA virus–60-70 nm in length with a genome of approximately 12 kilobase (kb) (Wahid et al., 2017). CHIKV is a mosquito transmitted alphavirus that is emerging as a global threat because of the highly debilitating nature of the associated disease and the unprecedented magnitude of its spread. Similar to the dengue virus (DENV), it originated in Africa and has since spread across the globe, causing large numbers of epidemics that have infected millions of people in Asia, the Indian subcontinent, Europe, the Americas, and the Pacific Islands. Similarly, dengue fever, incidence has grown in the last decade worldwide and according to the WHO, it has affected 3.9 billion people and in 128 countries are at risk of infection (Simo, Bigna, Kenmoe, et al., 2019). Approximately 390 million dengue infections occur each year in people worldwide; 96 million (25%) clinically manifest the disease, and 50% have mild disease (Bhatt et al. 2013). DENV is one of the most common arboviruses in tropical and subtropical mosquito bone viral diseases. DENV is transmitted by two main vectors, Aedes aegypti and Aedes albopictus . These vectors have become widely distributed across tropical and subtropical regions globally with the advent of global phenomena such as urbanization, high rate of population growth, inadequate water supply, sewers, poor waste management systems, and international travel. Although originally from Africa, Aedes aegypti has recently been found across the world, including Mexico, Central and South America, the Caribbean, and Asia. This mosquito is popularly called the yellow fever mosquito, whereas Aedes albopictus is known as the Asian tiger mosquito. Due to their invasive nature, arboviral diseases such as dengue, zika, yellow fever, and chikungunya are increasing global public health concerns due to their rapid geographical spread in their preferred eco-epidemiological zones. Knowledge of the contemporary distribution of their shared vectors, Aedes aegypti and Aedes albopictus , remains incomplete and is complicated by an ongoing range expansion fueled by increased global trade and travel (CDC, 2016; Kraemer et al., 2015) and climate change. There is a close relationship between ZIKV, DENV, CHIKV, and yellow fever virus (YFV), which currently causes a large outbreak in the Americas. Three genotypes were identified: West Africa, East Central South Africa, and Asia. Chikungunya disease is characterized by acute illness with fever >38.9 0 c severe polyarthralgia that can last for some years, and maculopapular pruritic rash lasting for a week. Infected neonates develop serious diseases that affect the heart, skin, and brain. Bleeding and disseminated intravascular coagulation have also been observed (Goeijenbier et al., 2016; Vu & Labeaud, 2018; Nanev Slavov & Kaori Otaguiri, 2015). In 2010, twenty-two countries in Africa reported sporadic cases or outbreaks of dengue; 12 other African countries reported dengue only in travelers. The presence of the disease and high prevalence of antibodies to dengue virus in limited serologic surveys suggest endemic dengue virus infection in all or many parts of Africa (Amarasinghe et al., 2011) . Malawi is one of the 13 countries in sub-Saharan Africa, which, according to Light Brown, shows that dengue has not been reported, but Ae . aegypti mosquitoes, a potential vector of ZIKV, DENV, CHIKV, and YFV, unlike Aedes albopictus , which has the same vectorial capacity as Aedes aegypti , studies have implicated Aedes aegypti in transmission as the most common vector (Amarasinghe et al., 2011). Studies carried out in Singapore have demonstrated the vectorial capacity of Aedes albopictus to transmit ZIKV since the virus was isolated in the salivary glands, which implies the potential to transmit the virus during blood meals (Wong et al., 2013). Similarly, a study carried out by Bisimwa et al. (2016) showed that mosquito vectors of arboviruses and their associated diseases of dengue, Chikungunya, and Zika were endemic and distributed throughout the Kyela district, Tanzania, which is a district neighboring the Karonga district in the present study area with varying densities, with vectors restricted to certain areas probably due to ecological and environmental adaptation. 1.2 Problem statement Arbovirus diseases are emerging health problems that require deliberate attention before they become a serious health problem. Factors that escalate the disease are related to vector breeding and increases in population numbers, such as increasing urbanization, poor urban planning, changes in climatic factors, and the availability of favorable micro-ecological conditions suitable for Aedes mosquito breeding in sub-Saharan Africa. In the face of such potential threats, there is a need for vigilance and establishment of preparedness measures before arboviral epidemics hit Malawi. Such an epidemic poses an overwhelming burden to the health system and potentially compromises the achievement of sustainable development goals (SDGs) (Baraka & Kweka, 2016). According to Higa et al. (2015), vector identification is crucial for controlling arboviral diseases, as the only way to reduce transmission. In Mozambique, cases were reported at the Eparse Islands in the Mozambique Channel in 2014, and several dengue cases were reported in the northern towns of Pemba, Nampula, and Mozambique. As Malawi is surrounded by countries with arboviral infection outbreaks, such as Tanzania and Mozambique, the likelihood of arbovirus transmission is very high. Since Karonga shares some characteristic geo-socio-ecological factors with neighboring countries like Tanzania and the rest of the southern region is bordered by Mozambique, the risk of transmission is very likely. In Malawi, a serological survey conducted in three districts, Blantyre (in the south), Lilongwe (in the central) and Karonga (in the northern region) established a prevalence of antibodies to these arboviruses which is indicative of previous infections. The prevalence of ZIKV antibodies was 0% in Blantyre, 0.6% (95%CI 0.0-1.7) in Lilongwe, and 2.9% (0.4-5.4) in Chilumba. The prevalence of DENV antibody was 2.2% (0.0-4.4), 5.6% (2.2-8.9) and 14.9% (9.6-20.3) at the three sites, respectively. The prevalence of CHIKV antibodies was 35% (27.6-42.4), 37.2% (30.1-44.4), and 67.2% (60.2-74.3%) at the three sites, respectively. This necessitated the need to carry out an entomological study to establish both the presence of Aedes mosquitoes and their importance in the transmission of arboviruses in the Karonga District. 1.3 Rational/Justification Despite the positive seroprevalence reported by some studies, Malawi has not yet established any surveillance system, developed any laboratory monitoring system, or any control program that deliberately targets the control of arboviruses such as ZIKV, DENV, and CHIKV. This could be because no deliberate effort has been made by the Ministry of Health (MoH) to develop control programs that aim to control these arboviruses. This may be because no large-scale studies have established the arboviral burden in the country at large. Some programs have been developed at the central level that target a few selected tropical diseases, such as malaria, schistosomiasis , and lymphatic filariasis , among many but not arboviruses. Similarly, the National Health Management Information System (HMIS) and Integrated Disease Surveillance and Response (IDSR) do not capture or report data on arboviruses in the country. The findings of this study will establish the species range of Aedes mosquitoes and the potential for arbovirus transmission in the country, and hence provide scientific evidence that can inform the MoH and all relevant stakeholders to institute programs aimed at the control of arboviruses and establish a robust surveillance and control program. 1.4 Hypothesis There are no infected arbovirus vectors present or distributed in the Karonga District in northern Malawi. 1.5 Objectives of the study 1.5.1 Main objective The main objective of this study was to determine the species abundance of Aedes mosquitoes and their importance in arbovirus disease transmission. 1.5.2 Specific objectives To determine the species abundance of Aedes mosquitoes in the Karonga district, northern Malawi. To assess whether the local Aedes mosquitoes found in the Karonga district in the northern region of Malawi carry arboviruses. CHAPTER TWO LITERATURE REVIEW 2.1 Arboviral mosquito vectors According to Wilkerson et al. (2015), the Aedes mosquitoes under study, belong to tribe Aedini , order Diptera and family Culicidae, genus Aedes , subgenus Stegomyia. The sculcllaris, albopictus, and aegypti subgroups. According to an earlier classification by Knight and Stones (1977) (Sivanathan, 2006), Aedes mosquitoes belong to Phylum Arthropoda; Class, Insecta; Order, Diptera; Family, Culicidae; Sub-family Culicinae; Genus, Aedes meaning unpleasant in Latin, also known as true flies, and it is sometimes further subdivided into subgenus Stegomyia. The Aedes mosquito species are Aedes aegypti and Aedes albopictus , from Africa and Asia, respectively. 2.2 Role of Aedes mosquitoes as arboviruses vectors Similar to most other flies in the order Diptera, Aedes mosquitoes have a life cycle that comprises eggs that hatch into adults having gone through metamorphosis to larva, pupa, and finally, adults emerge (Rozendaal, 1997). Aedes mosquitoes generally takes 7-10 days for adults to emerge from eggs, although eggs can survive for up to 8 months clinging to containers such as glue. This means that eliminating all larvae, pupae, and adult Ae. aegypti from a site, its population could recover two weeks later as a result of egg hatching following rainfall or the addition of water to containers harboring eggs. Aedes mosquitoes were distinct. They differ in terms of color, preferred habitat, biting habits, vectorial capacity, and breeding location (CDC, 2012a). Aedes aegypti is attracted to the chemical compounds emitted by mammals. These compounds included ammonia, carbon dioxide, lactic acid, and octenol. To date, studies done so far have shown no significant difference in terms of eggs laid in ovitraps with ordinary water or those with hay infusion and other substrates (Chadee, Lakhan, Ramdath, & Persad, 1993; Nazni et al., 2009). Viruses are transmitted to humans through the bites of an infective female Aedes mosquito, which mainly acquires the virus while feeding on the blood of an infected person. The life cycle of Aedes aegypti is entirely dependent on the environment created by humans. Larvae breed from a variety of artificial containers, such as jars, discarded cans, flower vases, cement tanks, ant traps, used tires, and plastic buckets around humans. Preference for humans as hosts is an important factor for transmission. Their close association with humans contributes largely to the effective transmission of arboviruses (Brady et al., 2014; Antonio, 2009). Many studies have implicated Aedes aegypti as a vector for the transmission of arboviruses, which have been proven to be competent vectors of arboviral diseases, such as dengue, chikungunya, yellow fever, and ZIKV (Calvez et al., 2016). However, it is now becoming interesting that some studies conducted recently in Africa implicate some species such as Aedes albopictus (Wong et al., 2013) and found that the vector was competent for ZIKV in the laboratory in Singapore. Furthermore, Diallo et al. (2014) found that ZIKV amplification was widespread in the Ke´dougou area; in Senegal, the team involved several mosquito species as probable vectors and encompassed all investigated land cover classes except indoor locations within villages. Dengue and Chikunguya have become major public health concerns for the past 50 years due to the rapid spread and increasing disease burden worldwide. In Iran, a recent study by Bakhshi et al. (2020) showed evidence of Chikungunya virus in mosquitoes sampled from some sites under study, In the United Kingdom (UK), Chapman et al. (2020), after a study of mosquitoes that were caught in the wild and infected with arboviruses showed vectorial capacity of several arboviruses. (Kraemer et al., 2015, Diagne et al., 2015, Powell & Tabachnick, 2013). In the Americas, confounding results were found in a study that aimed to evaluate the vector competence of mosquitoes. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), and South America (Brazil, French Guiana) for the currently circulating Asian genotype of arboviruses isolated from a patient in New Caledonia in April 2014. In the Pacific region, dengue and chikungunya were seen on the rise due to the presence of the competent Aedes mosquito. In Iran, (Chouin-Carneiro et al. (2016) and Calvez et al. (2016). In Nigeria, a study was carried out and established that not all febrile conditions present at the facility were malarial by displaying typical malaria symptoms. This is a similar observation in most of our hospitals, where most patients are characteristic of arboviral infection and not necessarily of malaria (Onyedibe et al., 2018). Another study on systematic measurement of head circumference at birth in Nigeria reported a prevalence of microcephaly (according to the WHO definition) of 10.6% in more than 3000 consecutive births in Lagos, Nigeria, in 2012 (Meda et al., 2016), establishing the presence of Zika virus. In Malawi, there are reports of Guillaine-Barre syndrome and microcephaly that are indicative of ZIKV infection among populations, and similar signs indicative of other arboviral infections. Further investigations are needed on the vector competence of other species associated with arboviral transmission to better understand the ecology and epidemiology of this virus. This makes the fight against arboviruses an upheaval task looking at emerging vector competencies, and needs to identify a variety of species ranges for vector competence studies. 2.3 Aedes aegypti (Linnae) The yellow fever mosquito, commonly known as Aedes aegypti (Figure 1), is a mosquito that spreads dengue fever, Chikungunya, Zika, and yellow fever viruses. This mosquito can be recognized by white markings on the legs and a marking in the form of a lyre on the thorax. The mosquito originated in Africa but is now found in tropical and subtropical regions throughout the world (Sivanathan, 2006). Aedes aegypti is a vector for the transmission of several tropical fevers. Only female blood bites are required to mature eggs. Although Ae. aegypti is known to be a susceptible host for dengue, as Aedes albopictus and Ae, polynesiensis , is, it is nevertheless a better vector, because it is a highly domestic species, which breeds in containers in or around houses, and with adults resting in homes. These characteristics ensure it has a much stronger vector-human contact than the other vectors (CDC, 2012b), this is also echoed by Service, 1992, There are distinct morphological differences between male and female Aedes mosquitoes. Males have feathery antennae. 2.4 Aedes albopictus (Skuse) The tiger mosquito or forest day mosquito Aedes albopictus (Stegomyia albopicta), from the mosquito (Culicidae) family, is characterized by black and white striped legs and small black and white striped bodies (Figure 2). It is native to the tropical and temperate regions of Asia. However, in the past couple of decades, this species has invaded many countries throughout the world through tire trade, and the global geographic distribution of Aedes albopictus has dramatically shifted as a result of the introduction of the species from Orient to New World, Europe, and Africa by frequent use of tires and recently due to uncontrolled urbanization in developing countries and global warming, which influence vector mosquitoes and exert an impact on vector-borne diseases. The frequent movement of people by aircraft has also resulted in the further introduction of vector mosquitoes to new places (Kraemer et al., 2015). This mosquito has become a significant vector in many communities because it is closely associated with humans (rather than living in wetlands), and typically flies and feeds in the daytime, in addition to at dusk and dawn. This insect is called a tiger mosquito because its striped appearance is similar to that of a tiger. Aedes albopictus is an epidemiologically important vector for the transmission of many viral pathogens, including West Nile virus, yellow fever virus, St. Louis encephalitis, dengue fever and Chikungunya fever. Unlike Aedes aegypti , Aedes albopictus eggs have the ability to diapause (the delay in development in response to regularly and recurring periods of adverse environmental conditions) during the winter season in temperate zone Asia. Gubler suggested that the maximum egg longevity period of Ae . albopictus was recorded for 243 d (Gubler, 1970). Aedes albopictus is a competent vector for many viruses, including dengue fever and Eastern equine en­cephalitis virus, and has been proven to be a competent vector for ZIKV (Wong et al., 2013). Its life cycle is closely associated with the human habitat, and it breeds in containers with standing water, often tires, or other containers. As a sylvatic (forest), it is a daytime feeder and can be found in shady areas where it rests in shrubs near the ground. Aedes albopictus feeding peaks in the early morning and late afternoon, and is an opportunistic and aggressive biter with a wide host range, including people and domestic and wild animals, unlike Aedes aegypti , which is mostly domestic both outdoors and indoors (Rios & Maruniak, 2014; Brady et al., 2014; Web Image, 2012). Ae. aegypti and Ae. Albopictus shares its life cycle and uses natural and artificial water-holding containers (e.g., tree holes, used tires, plastic containers, and clogged gutters) to lay eggs. After hatching, larvae grow and develop into pupae and subsequently into terrestrial, flying adult mosquitoes (CDC, 2016b). 2.5 Distribution of Aede s aegypti and Aedes albopictus A yellow fever mosquito ( Ae. aegypti ) has a cosmo-tropical distribution and spreads to more temperate regions during the summer months. Originating in Africa, the Ae. aegypti is now present globally in the tropical and subtropical regions. Research on Aedes mosquitoes over the past decade has documented two morphologically distinct subspecies of Aedes aegypti : Ae . aegypti aegypti and Ae . aegypti formosus (Mattingly 1967). Knowledge of the contemporary distribution of their shared vectors, Aedes aegypti and Aedes albopictus , remains incomplete and complicated by an ongoing range expansion fueled by increased global trade and travel. Mapping the global distribution of these vectors and the geographical determinants of their ranges are essential for public health planning. Today, about half of the world’s population is at risk of dengue infection, and chikungunya outbreaks, which were previously limited to Africa and Asia, have recently been reported in the Caribbean, South America, and Europe. Dengue and chikungunya viruses are transmitted between people by two mosquito species, Aedes aegypti and Ae. albopictus . Therefore, it is important to identify areas at risk where these mosquito species are found around the globe. It is also important to predict where these species could become established if they were introduced to identify areas that could become at risk in the future (Kraemer et al., 2015), as shown in the maps below: A yellow fever mosquito ( Ae. aegypti ) has a cosmo-tropical distribution and spreads to more temperate regions during the summer months. Originating in Africa, the Ae. Aegypti is now present globally in the tropical and subtropical regions. Research on Aedes mosquitoes over the past decade has documented two morphologically distinct subspecies of Aedes aegypti : Ae . aegypti aegypti and Ae . aegypti formosus . The two subspecies were distinguished based on the color of the tegument and abdominal scale patterns. The arrival of Aedes albopictus has been correlated with a decline in the abundance and distribution of the yellow fever mosquito Aedes aegypti (Linnaeus). Aedes albopictus larvae outcompete Ae. aegypti larvae for food, and develop at a faster rate ( Barrera 1996 Competition and Resistance to Starvation in Larvae of Container-Inhabiting Aedes Mosquitoes Ecol Entomol 21 117-127.Pdf , n.d.). From Figure 4, which shows the predicted distribution of Aedes albopictus , Malawi still falls within the region with the median probability of the vector distribution. Thus, the country established itself in terms of surveillance and response activities. 2.6. Arboviruses Arboviruses are known to affect communities globally, and some have reached the extent of outbreaks. Arbovirus means ar thropod- bo rne vi ruses. The word arthropod comes from the Greek root words arthro - meaning joint and - pod meaning foot and that refers to a unique feature of the group of insect vectors. Jointed legs, also called appendages, vary widely in number and function. Appendages were used for eating, feeling, sensing, mating, respiration, walking, and defense. Arboviruses are categorized into five families: Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, and Rhabdoviridae. Arboviruses vary in their size and shape. Most are spherical, except for Rhabdoviridae, which is bullet-shaped. Spherical viruses exhibit icosahedral symmetry. Virions range in size from approximately 45 nm in diameter (flaviviruses) to > 380 nm in length (some rhabdoviruses). The genomes of almost all the arboviruses contain RNA. RNA may be single- or double-stranded, linear or circular, and may be positive or negative. Positive-sense RNA can act as mRNA, and genomes with this type of RNA are termed infectious. On the other hand, genomes with negative-sense RNA must first make positive-sense RNA for transcription and are termed noninfectious (Simpson, 1972). These arboviruses are notable for causing serious diseases such as dengue, Zika, and Chikungunya, which sometimes occur during an outbreak and cause mortality in society. This study focused mainly on dengue and chikungunya. Chikungunya virus belongs to the genus Alphaviruses and family Togaviridae, which are of medical importance because they have caused major outbreaks worldwide. This study focuses mainly on dengue and Chikungunya 2.6.1 Dengue virus Dengue virus falls under the Genus Flavirus and family Flaviviridae, similar to Zika Virus. The presence of the vector and the diagnosed disease in some countries has encouraged us to develop systems for entomological surveillance and response. Most parts of Africa have recorded the presence of the Aedes mosquito, although others have not yet. A systematic review by Simo et al. (2019) noted that although a total of 22 countries in Africa reported sporadic cases of outbreaks of dengue fever, to date, no study has accurately investigated the epidemiology of DENV infection among febrile and apparently healthy populations in this continent and thus need to do so. As shown in Figure 5, dengue was reported among travelers in 34 countries in which dengue has been reported, including dengue reported only in travelers and Ae . aegypti mosquitoes as shown by the brown colour indices. Light brown indicates 13 countries (Mauritania, The Gambia, Guinea-Bissau, Guinea, Sierra Leone, Liberia, Niger, Chad, Central African Republic, Republic of the Congo, Malawi, Zimbabwe, and Botswana) in which dengue has not been reported but that have Ae. aegypti mosquitoes. White indicates five countries (Western Sahara, Morocco, Algeria, Tunisia, and Libya) for which data on dengue and Ae. aegypti mosquitoes were unavailable (Were 2012). 2.6.2 Chikungunya Virus Unlike DENV, CHIKV is widely isolated in Africa and the rest of the world (Figure 6). Contemporaneous systematic meta-analysis prevalence study by F. B.N. Simo et al., 2019, established a high prevalence of CHIKV infection in Africa, especially during outbreak periods. Chikungunya disease has been identified in nearly 40 countries, including half of them in Africa. Although endemic to Asia and Africa, the virus has invaded new territories, including the Indian Ocean island and Italy. This has made it a public health concern, and hence the need for awareness and response (CDC & PAHO, 2011). Based on the chronology of the Chikungunya outbreak, Wahid et al. (2017) In Africa, CHIKV was first reported in Tanzania in 1952. This was followed by several other epidemics in the Central African Republic, Guinea, Burundi, Angola, Uganda, Malawi, Nigeria, Democratic Republic of the Congo, and several other states. From the 1960s to the 1990s, outbreaks were recorded in the Democratic Republic of the Congo, Central African Republic, Malawi, Uganda, Burundi, Angola, Guinea, South Africa, and Nigeria. Almost half of the cases were reported in June 2004 during an outbreak that occurred in Lamu Atoll, Kenya. The first outbreak in Malawi occurred between 1987–89. A few cases have been reported in 2001 and 2015. As of October 30, 2020, Malawi has been in countries where Chikungunya has been reported (CDC, 2016a). Malawi falls in countries where chikungunya has been reported; however, few studies have been conducted to ascertain the extent of the outbreak. The Biology Department of Chancellor College carried out a Risk Map for various vectors and their associated diseases being transmitted. Karonga was marked as an area at high risk of arboviruses (Figure 7). The map showed that Karonga and other districts were at the highest risk of arboviruses. R arbovir= p (imm+dens) + β (env temp/alti+precip) + α ( vecden+ type+ c ........ 2.7 Life cycle of arboviruses Arboviruses are maintained in nature through biological transmission between susceptible vertebrate hosts by blood-feeding arthropods (mosquitoes, psychodids, ceratopogonids, and ticks). Vertebrate infection occurs when the infected arthropod takes a blood meal. The term 'arbovirus' has no taxonomic significance (States, 2007). Arboviruses have several types of life cycles, but many have a sylvatic cycle, such as Aedes albopictus , while some also have an urban cycle, the sylvatic cycle (sometimes known as the jungle cycle). The viral cycles between an arthropod and a mammalian host with humans are usually a dead-end host infected by the arthropod and urban cycle. The virus cycles between humans and an arthropod species. There is an urban cycle for yellow fever and dengue fever (both also have a sylvatic/jungle cycle). If there is an urban cycle, using window screens, bed nets, etc., to prevent access of mosquitoes to viremic patients may reduce transmission (Ayres, 2016). Arboviruses are mainly transmitted by Aedes mosquitoes and are widespread in both urban and peri-urban areas. Zoonotic relationships are known to occur between humans and non-human primates. The role of other reservoirs in sexual transmission remains unclear. CHAPTER THREE METHODOLOGY 3.1 Study Area The study was carried out in the Karonga district, which is located in northern Malawi between the latitudes and longitudes of -10.362124 and 34.206370, respectively. Karonga District is bordered by Chitipa District to the West and Rumphi District to the South (Figure 10). The district headquarters is located approximately 50 km south of the Tanzanian border and 585 km north of the capital city of Malawi, Lilongwe. The total land area of the district is 3355 square kilometers making for 3.5% of the total land area of Malawi (94276 sq km). 3.2 Topography and Climate (Council, 2011) The relief of the district has a wide range of 400m at the lakeshore and 2400m at the high Nyika Plateau. It appears predominantly hilly, going westward from the lake. The most outstanding landforms can be divided into three zones: a) High Hills and Plateau Zone, b) Rift Valley Escarpment Zone, and c) Lakeshore Plain Zone. Similar to other districts in the country, Karonga has a subtropical climate. 3.3 Study Design This is a cross-sectional study. Sampling followed a transect design from south to north of the district, with six sampling points established. Sampling was carried out once a week over a period of three months. The location of the study was selected using existing health clusters, viz: Hara, Karonga Boma, Kayelekera, Iponga, and Kaporo, and simple random sampling was performed where all areas under the cluster had an equal chance of being selected. Simple random selection was performed for all villages under the cluster to determine the placement of traps in villages from the clusters that met the criteria for the possible habitation of Aedes mosquitoes. A single location was determined, and traps were placed at the sampled locations in each cluster (Figure 10). 3.4 Weather data Weather data were obtained from the Metrological Department during the study period from January 2018 to December 2018. Table 1: Weather data for Karonga District Weather data for Karonga district – 2018 Year Month Rainfall Max_Temp Min_Temp Windspeed 2018 Jan 180.7 30.3 21.2 1.4 2018 Feb 124.6 31.0 21.7 1.1 2018 Mar 219.1 30.2 16.2 1.1 2018 Apr 234.5 29.5 20.1 1.2 2018 May 0.0 29.8 19.5 1.8 2018 Jun 0.0 28.6 17.7 1.9 2018 Jul 1.5 28.1 16.1 2.3 2018 Aug 0.0 29.8 16.5 1.7 2018 Sep 0.0 31.9 19.2 2.0 2018 Oct 0.0 32.7 21.3 2.3 2018 Nov 8.0 33.5 22.7 2.0 2018 Dec 198.7 31.5 22.1 1.7 Source: Metrological department. Blantyre, Malawi 3.5 Mosquito trapping and sampling 3.5.1 Sampling of mosquitoes catching sites In this study, sampling targeted all life cycle stages of Aedes mosquitoes (larvae, pupae, and adults) present in the selected communities. 3.5.2 Mosquito trapping Ovitraps were distributed in the selected transect points in the urban (Karonga town at Mwangwambila village, and Kaporo at Mwenengolongo and Kasebe) and rural ( Hara at Hangalawe, Iponga at Weleweta and Kayelekera at Sere village) areas of Karonga district (Figure 10). Ovitraps were deployed for one week, and on the seventh day, adult and aquatic mosquito stages were sampled from traps. This routine was followed to avoid production of the second-generation mosquito family. Once the adult mosquitoes laid eggs in the traps, they were trapped inside, and both emerged adults (F 1 ) and first-generation adults (F 0 ) were collected. In addition, mosquito larvae were collected using dippers from artificial breeding habitats such as discarded tyres, troughs, holes, and other containers. Larval samples were placed in small containers and placed in rearing cages until adults emerged. After emergence, the adult mosquitoes were collected using a pen siphon, and each was placed in its own Eppendorf tube labeled according to site and placed in a-80 0 C refrigerator. Mosquitoes were transported using liquid nitrogen from Karonga to the laboratory at Chancellor College (now the University of Malawi). Using a dissecting microscope, Aedes mosquitoes were morphologically identified using a mosquito key (BUXTON, 1941) and separated from other types of mosquitoes (Kean et al., 2015). Because arboviruses are vertically transmitted, all Aedes mosquitoes were preserved in 90% ethanol and stored at -80°C for further analysis. 3.5.6 Mosquito Data Collection Mosquito data Mosquito ovitraps and larval rearing provided tangible mosquito counts at each study site. Table 2: Showing Mosquito collection sites and the number of ovitraps deployed for Karonga District Health cluster Short code Village name Number of ovitraps Hara 2HA Hangalawe 7 Iponga 4IP Gweleweta 12 Kaporo 5PK Mwenengolondo and Kasebe 12 Karonga Town 1KA Mwangwambila 20 Kayelekela 3KY Sere 3 Total 54 3.6 Mosquito processing and analysis 3.6.1 Isolation of RNA A Qiagen RNA extraction kit was used for RNA processing (Santos et al., 2004). Twenty mosquitoes from each site were pooled into 2 ml a single centrifuge tube as pooling method for all sites (Table 2). For each site, 500 µL of medium was added, ground using a pestle with a pestle homogenizer, and manually homogenized. The medium was centrifuged at 6,000xg (8,000 rpm) for 10 min. A total of 140 µL of the specimen was removed for RNA extraction and the remaining mixture was transferred to a screw-cup tube. The screw cup tube was labeled and the pestle was washed by rinsing in Jik. The stored homogenate RNA carrier was in a-80 0 C freezer. Homogenate carrier RNA was stored and subjected to RNA extraction, which included lysis, washing, elution, and extraction of pure RNA. Carrier RNA was added to buffer AVL. A buffer (310 µL of AVE buffer was added to a tube containing the lyophilized carrier RNA. Mixing was performed by inverting the tube ten times to reconstitute the carrier RNA. A total of 310 µL of dissolved carrier RNA was transferred to the buffer AVL. For six samples, the net was 33.6 µl which was stored in a refrigerator. A total of 560 µL of buffer AVL with carrier RNA was pipetted into a 1.5 ml centrifuge tube. A total of 140 µL of specimen was added to the mixture, pulse vortexed for 15 s, and then stored for 10 min at room temperature. The mixture was briefly centrifuged for 10 s, 560 µL of ethanol was added to the sample, vortexed for 15 s, and briefly centrifuged. The solution mixture (630 µL) was added to a QIAamp spin column (2 ml without wetting the lid and closing the cap. The mixture was centrifuged at 6,000 x g and 8,000 rpm for 1 min. The QIAamp spin column was placed into a clean 2 ml collection tube, and the tube containing the filtrate was discarded. The preceding process was repeated, the QIAamp spin column was carefully opened, and 500 µL of buffer AWI was added. The mixture was centrifuged at 8,000 rpm for 1 min. The QIAamp spin column was placed in a clean 2 ml collection tube, and the collection tube containing the filtrate was discharged. The QIAamp was carefully opened, and 500 µL of buffer AW2 was added, cap closed, and centrifuged at 13,500 rpm for 3 min. QIAamp was placed in new 2 ml centrifuge tubes, and the old filtrate was discarded. Sixty microliters of AVE buffer were added to the center of the column, and the cap was closed. The mixture was incubated at room temperature for 1 min and then centrifuged at 8,000 rpm for 1 min. The final eluate contained 90% of RNA. Pure RNA was stored in a 2 ml centrifuge tube at -80 0 C freeze. 3.6.2 Polymerase Chain Reaction- Reverse Transcription (RT-PCR) This converts single-stranded RNA into complementary DNA (cDNA) for PCR. This reaction involved reverse transcription, denaturation, annealing, and extension. The RT-PCR reagents were put into one master mix tube totaling to 95µl and centrifuged. This comprised 4µl 5X RT buffer, 2µl dNTP, 0.5µl cFD2 as forward primer, 1µl RT enzyme polymerase, 11.5µl PCR water, 1µl template RNA. For five sites, the total was 95µl and each site was 20µl. The thermocycler was programmed in three steps as follows: One cycle for 42 0 C for 20 minutes, then 99 0 C for 5 min and 4 0 C for ∞ The processes above complete the reverse transcription and was done for both dengue and Chikungunya. The final product produced was cDNA (Domingo & Patel, 2012, Petz et al., 2014) 3.6.3 Polymerase Chain Reaction (PCR) The master mix for the PCR assay was prepared totaling volume of 97.5µl. Extracted pure RNA was added to each tube for analysis. There were five tubes each representing a sentinel site for mosquito sample traps or rearing. For dengue and Chikungunya, the mix was as follows: 4µl 5X RT buffer, 1µl dNTP, 0.5µl dengue 802 as forward primer for dengue and 0.5µl cFD2+MAMD as reverse primer for dengue; 0.5µl CHIKVFLY as forward primer for CHIKVFLY and 0.5µl cFD2+MAMD as reverse primer for CHIKVFLY; 0.5µl CHIKV E1 as forward primer for CHIKV E1 and 0.5µl cFD2+MAMD as reverse primer for CHIKV E1, 0.25µl polymerase, 12.75µl PCR water, 0.5µl template RNA. The reaction tube was 20µl. This was performed for each primer for dengue, chikungunya, CHIKV FLY, and CHIKV E1 (Scaramozzino et al., 2001; Lee et al., 2012). Distributed the 19.5µl master mix was distributed into each tube for the five sites using short codes denoted at each site. The tubes were capped and vortexed to mix reagents. The thermocycler run for each was programmed as follows: For CHIKV E1 and FLY The steps are as follows. 94 0 C for 3 min for one cycle. Then for 30 cycles at: 98 0 C for 20 sec 57 0 C for 20 sec 72 0 C for 40 sec 4 0 C for ∞ For dengue The steps are as follows. 94 0 C for 3 min for one cycle. Then for 30 cycles at: 98 0 C for 20 sec 60 0 C for 20 sec 72 0 C for 1 min 4 0 C for ∞ The PCR tubes were loaded in a thermocycler, the lid was closed, and the PCR program was run as indicated for each arbovirus, as described above. 3.6.4 Gel electrophoresis It is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. The gel electrophoresis apparatus consists of a gel, often made from agar or polyacrylamide, and an electrophoretic chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end and an anode (positive terminal) at the opposite end. An electric current was applied to move the molecules to be separated through a gel. Pores in the gel work like sieves, allowing smaller molecules to move faster than larger molecules. In this study, DNA was separated using agarose gel electrophoresis, where the DNA was loaded into pre-cast wells made of a gel comb, and an electric current was applied through the apparatus. The phosphate backbone of the DNA (and RNA) molecule is negatively charged; therefore, when placed in an electric field, the DNA fragments migrate to the positively charged anode. Since DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight. The gel was prepared using a 2% agarose gel with 1X TAE buffer as an electrolyte in a charged electric field. For 2% agarose gel preparation, we measured 2 g of agarose in an Erlenmeyer flask containing 75 ml 1x TBE buffer. Agarose (1.5 g) was weighed, put in 1X TAE buffer, and mixed. The flask was covered with Kimwipes and heated in a microwave until the agarose was dissolved for two minutes. It was left to cool to approximately 60°C on the bench for several minutes, so the agarose should not solidify. The agarose solution was stained with 5 µL GelGreen Nucleic Acid 10000 × in water. The mixture was prepared by swirling a flask. Agarose was poured into the mold (75 ml and 25 ml at room temperature to a gel thickness of 3-5 mm. The comb was placed on top of the trays and was carefully removed after 30 min at room temperature. The gel was then positioned in an electrophoresis tank. A 1X buffer was added as an electrolyte to cover the gel to a depth of approximately 5 mm. The wells of the gel were loaded with a mixture of 8µl of DNA samples or PCR products and with loading dye 2µl bromophenol blue in each tube to a total mixture of 10µl using pipettes slowly. Each slot represents a sample from a designated capture site. Gel wells were filled with GeneLadder 100, negative control (PCR water), negative control with polymerase, positive control with CHIKV E1, Samples 1KA, 2HA, 3KY, 4IP and 5KP representing catching sites, then well with samples for the sites for CHIKV Fly. For Chikungunya, both samples were run on the same gel. The dengue gel was run separately. Electrical leads were switched on, and DNA in the gel moved towards the anode (red lead) after the application of a voltage of 1-5V/cm. The gel was run until the gel-loading buffer stain migrated to the appropriate distance (normally, until the bromophenol blue dye front migrated ¾ of the way down the gel). The current was turned off and the leads were removed. The gel was examined for dengue and chikungunya separately, and the observed bands were observed in the ultraviolet transilluminator gel green and photographed, as shown in the results section in this write-up (Lee et al., 2012). 3.7 Data management and analysis Data were collected using tablets; each data point was coded, and coordinates were obtained using a global positioning system [GPS]. Data were collected using the Open Data Kit (ODK), which was installed in tablets for the collection of vital data elements needed for analysis. Data were analyzed using Microsoft Excel to generate descriptive statistics. for the weather and mosquito data per site, respectively. The results are presented as graphs and tables, with the corresponding proportions. 3.8 Ethical considerations The study did not involve household surveys or interviews; hence, sample collection did not interfere with human activity. This study was a continuation of the human study for arboviral serological study in people who already had ethical approval through the College of Medicine and Malaria Alert Centre. 3.9 Study limitations The study analyzed all potential arboviruses likely to be present in the study region, including Zika, but the availability of reagents was a limiting factor. Only DENV and CHIKV were investigated using the available primers. Lengthy procurement processes are challenging. Molecular reagents are not internally sourced; therefore, it takes much longer to procure them. Consequently, the samples took much longer to analyze and, in the process, some stored samples degraded, resulting in non-amplification (null scoring). Another factor could be poor coordination and communication between the collaborators as the collaborators involved Malaria Alert Centre, MEIRU, and Chancellor College. CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Types of mosquitoes in Karonga District 4.1.1 Mosquito species collected in Karonga District A total of 1,100 mosquitoes were collected across the six study sites, with 578 (53%) belonging to the other culicines and 522 (47%) Aedes aegypti (Table 3). No Ae. albopictus (0.0%) was detected across the sampling sites. Of the total mosquitoes collected, 249 (23%) were originally collected as larvae from discarded tyres or troughs, compared to 861 (77%) adults collected using ovitraps. Ovitraps were more successful at sampling Aedes mosquitoes than larval sampling. However, only two places had both methods of collection, viz, Kaporo and Karonga, yielding 49% and 51% of Aedes mosquitoes through tire collection. Table 3 shows the mosquito yield per site and their corresponding proportions. Table 3: A summary of Aedes spp, culicines, and their proportions recorded per site Site name Aedes aegypti % Aedes albopictus % Other culicines % Hara 82 15.7 0 0 222 38.4 Iponga 144 27.6 0 0 48 8.3 Kaporo 165 31.6 0 0 73 12.6 Karonga Town 128 24.5 0 0 128 22.1 Kayelekera 3 0.6 0 0 107 18.5 Total 522 100 0 0 578 100 4.1.2 Aedes mosquito transect distribution across Karonga District Kaporo yielded the majority of Ae. aegypti mosquitoes (31.6%), followed by Iponga (27.6%), Karonga Town (24.5%), Hara (15.7%), and Kayelekera (0.6%) had the least mosquitoes. This means that the population densities of Aedes aegypti mosquitoes decreased from the farther north as you went to the southern part of the district (Figure 10). 4.1.3. Distribution of Aedes mosquito densities per month In the first month of March, only one Ae. aegypti mosquito, representing 0.19% (n=522) from Hara, making it the month with least mosquito yield of mosquitoes. The highest Aedes mosquito yield was recorded in April (81.23%), followed by May (18.58%). Kaporo registered the highest yield (32%) at all sites (Figure 11). The rainfall season was from December to May. Heavy rainfall was recorded in April (234.5 millimeters rainfall, followed by March receiving 219.1 millimeters with May (0 mm). During the peak month of the rainy season in April, all sites recorded high mosquito densities, except for Hara. However, Hara recoded the highest number of mosquito densities in May where there was no rainfall recorded than any other site (45/97) representing 46%, followed by Iponga (41/97) representing 42%, while Kaporo and Kayerekera had zero mosquito respectively in the same month. Kaporo, which had the highest percentage of Aedes aegypti mosquito (32%) than any other site, recorded such a yield in April, which also recorded the highest rainfall in the three months of the study. There was a sharp decline in mosquito densities at all sites in May, which did not record any rainfall. Kayelekera recorded only three mosquitoes during the study period, which was also recorded in May. 4.2 Presence of arboviruses in the vector Aedes aegypti mosquitoes The final results of the laboratory processes described in sections 3.4.3, 3.4.4, and 3.4.5, which highlight the laboratory analysis of RNA isolation, reverse transcription PCR, PCR, and viewing in the ultraviolet transilluminator Gelgreen separately of the Aedes aegypti mosquito sample, showed the presence of both Chikungunya and dengue arboviruses as reagents used to detect these viruses. All mosquito catching sites had mosquitoes that were infected with these arboviruses (Table 4). Table 4: Presence of arboviruses in mosquitoes per site across Karonga district in northern Malawi Health cluster Site name Number of ovitraps Number of Aedes mosquitoes Presence of arboviruses (CHIKV and DENV) Hara Hangalawe 7 82 Yes Iponga Gweleweta 12 144 Yes Kaporo Mwenengolondo and Kasebe 12 165 Yes Karonga Town Mwangwambila 20 128 Yes Kayelekela Sere 3 3 Yes Total 54 522 Each site showed visible bands after electrophoresis for both the chikungunya virus and dengue virus. 4.3 Discussion 4.3.1 Types of Mosquitoes in Karonga District Aedes aegypti was present in all traps throughout the district, indicating that it was widespread. These vectors has been proven to be globally distributed (Diagne et al., 2015, Kraemer et al., 2015, Simard et al., 2005, Farajollahi & Nelder, 2009). The study observed that Aedes mosquitoes were inversely distributed to other culicines ( Culex sp. and Mansonia sp. ). A nationwide study carried out from November 2011 to April 2012 by Maekawa et al. (2021) established the presence of these Culex complexes across the country, and the species was confirmed to be spread across Africa according to some studies. Interestingly, the urban town of Karonga recorded almost equal numbers of known vectors, Aedes aegypti , and other culicines (50%). Kayerekera Mine yielded the highest percentage (97%) of culicines (107) compared to the known vector Aedes aegypti (3%) and had the least number of Aedes aegypti than all study areas. This is in contrast with another entomological study carried out to determine risk factors for anopheline in rural and urban areas in Blantyre by Mzilahowa et al. (2016), who found that rural areas had a higher risk than urban areas. However, migration factors have been attributed to malaria-related morbidity due to malaria in urban areas. Study sites that yielded the highest numbers of Aedes aegypti mosquitoes, such as Kaporo and Iponga, yielded the lowest numbers of other culicines, 12.6% and 8.3%, respectively, compared with sites that yielded fewer Aedes aegypti , such as Hara, Kayelekera, and Karonga, which yielded 38.4%, 18.5%, and 12.6%, respectively, indicating differences in the presence of breeding habitats. The presence of suitable breeding habitats might partly explain the distribution of Aedes aegypti and Culicines along the sampling sites. Similar studies conducted in Kinondoni district and northern Tanzania in Kilimanjaro by Hertz et al. (2016) and Ngingo et al. (2022) found similar results, with only Aedes aegypti found in the study area. On the contrary, studies conducted in Senegal (Diagne et al., 2015) have shown that the Aedes albopictus vector is a competent vector to transmit both dengue and Chikungunya, even though Aedes aegypti stands out as the best competent vector. In Mozambique, a survey in 32 urban areas similarly found that only 0.03% of the total samples collected from 2,807 were Aedes albopictus , thus corroborating the findings of this study, which did not find Aedes albopictus (Abílio et al., 2018). Information from the vector control unit at the Kayelekera mine site indicated that the area underwent vector control to stop mosquito breeding, showing that mosquito control is effective against Aedes aegypti and not against other culicines that are prevalent in the area. 4.3.2 Distribution of Aedes Mosquitoes in Karonga District Mweya et al., 2013 and Bisimwa et al., 2016 established presence of both mosquito vector densities as well as presence of arboviruses in the vectors in their study in Mbeya and Kyera districts in Tanzania which lies to the north of Karonga district, Malawi. Kaporo and Iponga, which are in the northern part, share almost the same geographical and climatic features as the neighboring districts in Tanzania; in this study, these sites registered more mosquitoes than other sites in the south. This may indicate a trend of mosquitoes in motion, as Aedes mosquitoes can easily be transported in moving objects, containers, and even cars and pose a risk of spreading to non-habitable areas. In Spain, the Peruvian Amazon, and Ethiopia, different studies carried out by different researchers confirmed the dispersion of Aedes albopictus and Aedes aegypti and confirmed that vehicles such as cars and boats in addition to containers were responsible for the dispersion of these arboviral vector mosquitoes (Eritja et al., 2017, Getachew et al., 2015; Guagliardo et al., 2015). This is proof of vector dynamics due to many geosocial and anthropometric factors, and could be one of the possible factors of Aedes aegypti spread throughout the district. The presence of Aedes mosquitoes alone ascertains the potential for arboviral transmission according to Weetman et al. (2018), who found that Aedes mosquitoes were implicated in arbovirus transmission more than other species. However, in Iran, Bakhshi et al. (2020) found evidence of some pooled 6 samples from non-Aedes sp. , which were positive for the Chikungunya Asian type. This however, does not mean that some of these vectors have the competency to transmit the arboviruses as efficient as Aedes aegypti which is a known competent vector or arboviruses (Bisimwa et al., 2016; Chapman et al., 2020 and Calvez et al. 2016). Similar to Tanzania and Mozambique, which are neighboring countries, Aedes aegypti was found in large numbers and caused outbreaks in the respective areas and throughout the country (Higa et al., 2015, Mweya et al., 2013). This could be true for our country since the presence of Aedes aegypti throughout the district of Karonga would seriously put people at risk, as Karonga borders Tanzania and Zambia. Suspected outbreaks of chikungunya and dengue outbreaks in Zambezia Province necessitated a nationwide survey as people were presenting at facilities with arboviral infections. According to the findings of the study by Mugabe et al., 2018, the patients were positive to Chikungunya and dengue and the abundance of the Aedes aegypti throughout Mozambique, entails a great risk to Malawi as the majority of Malawi’s southern part is bordering Mozambique. An unpublished report by the Biology Department at the University of Malawi, Chancellor College by Pemba et al.. established a risk map based on vector composition, and Karonga was marked as a high-risk area for arboviral transmission (Figure 7). Therefore, Malawi should be vigilant and should institute a nationwide survey to determine the presence of arboviruses in mosquitoes. 4.3.3 Seasonal distribution of Aedes mosquito in Karonga district, northern Malawi Seasonal changes and temperature had a direct impact on the abundance of Aedes aegypti. This study found more Aedes aegypti during the month of April and towards the end of the rain, with March and April recording 219.1 mm and 234.5 mm respectively. The same month of April recorded 29.5 0 C and March 30.2 0 C and thus the range was favourable for Aedes mosquitoes and provided optimal breeding. Akram et al., 2009, in his study on seasonal distribution and species of daytime biting mosquitoes found that Aedes mosquitoes population density rapidly increased by 26.3% following the rainy season in July with temperatures between 38 0 C-42 0 C. However, in the same study, he found a population density decline in mosquitoes when the temperature reached approximately 45 to 50 0 C. Our results are consistent with findings from Yaoundé Cameroon, Djiappi-tchamen et al. (2021), where the abundance and distribution of Aedes mosquito species in each ecological setting was significantly different between the dry and rainy seasons (p < 0.0001). A higher density of Aedes mosquito species was observed during the rainy season (n = 4706; 74.32%) than in the dry season (n = 1626; 25.67%), especially in peri-urban (93.83%) and urban areas (96.81%). This implies that when active monitoring of arbovirus vectors is enforced, seasonal surveillance must be considered as the densities of mosquitoes vary with varying seasonal factors. 4.3.4 Presence of arboviruses in the mosquitoes All sentinel sites for Karonga showed visible white bands during agarose gel electrophoresis, as all bands were along the positive control after reverse transcription PCR (RT-PCR) for both Chikungunya and Dengue. All sites were sensitivity to CHIKV E1 recombinant and not CHIKV FLY, which means that all the sites in Karonga had Chikungunya virus class E1, not the CHIKV fly, as both primers were used (Mavale et al., 2012; Cho et al., 2008). However, similar studies were carried out by Joannides et al., 2021, who wanted to find species composition and risk of arbovirus transmission in some parts of northern Ghana, and did not yield any positive results for arboviruses after RT.PCT from the 75 pools of Aedes mosquitoes. The findings of this study are, however, more consistent with those from the Kyera district, which is to the north of the Karonga district. In their findings from an entomological study carried out in Kyera town, Kajunjumele, Ipida, Matema, and Njisi villages from April to June 2015 (almost the same months as our study), Bisimwa et al., 2016; Bisimwa, N P, Angwenyi, S, Kinimi, E, et al., 2018 found arboviruses in 24 pools ofAedes mosquitoes from 480 Aedes mosquitoes collected. Arboviruses were detected in nine pools (37.5%), including alphaviruses (eight pools) and flaviviruses (one pool). None of the samples were positive for bunyaviruses. Chikungunya virus (CHIKV) was detected in six (75%) alphavirus-positive pools that were collected mostly in areas where rice cultivation was common. However, this study focused only on Dengue and Chikungunya, and all site samples were positive for flavivirus (CHIKV) and alphavirus (DENV). The findings of this study may indicate that people from these areas are equally exposed and at risk of arboviral transmission, similar to their counterparts in the neighboring district in Tanzania. This directly correlates with the human serum survey that isolated both chikungunya and dengue in patients who presented with febrile conditions at the facility in Karonga, Lilongwe, and Blantyre districts. CHAPTER FIVE CONCLUSION AND RECOMMENDATION 5.1 Conclusions This study confirms the presence of the Aedes aegypti mosquito, which is a known vector for arboviruses that cause dengue fever and Chikungunya disease. Due to limitations in reagents, the study has only isolated dengue and chikungunya, but the presence of the competent vector shows that the population is also at risk of other arboviruses such as Zika. This study further complemented the human sero study that already established the presence of arboviruses in the human serum; thus, it has linked the transmission to humans due to infected vectors. 5.2 Recommendations There is a need for both habitats for breeding of these vectors must be taken care of if we are to control the spread of arboviruses. However, the other weather parameters were not critically analyzed and can be recommended for further studies to ascertain where rainfall and other weather parameters have a direct impact on the abundance of mosquitoes. Another area for further study could be the link between breeding sites and the abundance of different species. I would recommend that further studies be carried out on the linkage between the vectors and human serology throughout the district of Karonga, and institute further serological studies as well as entomological studies that will further describe the disease burden in the district and immediately institute some preventive measures for the district. These findings would be shared with the Public Health Institute of Malawi (PHIM) and all concerned stakeholders to trigger the establishment of monitoring and surveillance programs on arboviruses before the district and country experience outbreaks of dengue and chikungunya, which have debilitating effects on the health and economy of the country. I would recommend more sentinel surveillance sites to be established throughout the country to establish the burden in the Karonga Abbreviations CDC: Centers for Disease Control and Prevention CHIKV: Chikungunya Virus DENV: Dengue Virus GIS: Geographical Information System HMIS: Health Management Information System IDSR: Integrated Disease Surveillance and Response ITCZ: Intertropical Convergence Zone MEIRU: Malawi Epidemiology and Intervention Research Unit MLW: Malawi-Liverpool Welcome Trust Clinical Research Programme ODK: Open Data Kit PCR: Polymerase Chain Reaction QGIS: Quantum Geographical Information System RNA: Ribonucleic Acid SDGs: Sustainable Development Goals WHO: World Health Organization ZIKV: Zika Virus Declarations I, the undersigned, hereby declare that this thesis is my original work which has not been submitted to any other institution for similar purposes. Where other people’s work has been used acknowledgements have been made as reference in the document. DEDICATION To my family members who endured my absence during my study and to all those who have dedicated their lives to medical entomology. ACKNOWLEDGEMENTS I sincerely thank my supervisors, Associate Professor Dylo Pemba and Dr. Themba Mzilahowa, for their effort, valuable time, guidance, and mentorship. In addition, I would like to thank Dr. Steve Gowero for providing the much-needed guidance during proposal development and laboratory work at Chancellor College. Special gratitude should go to Dr. Chigwechoka and Mr. Yohane Kazembe for dedicating their precious time to helping me during laboratory analysis. I am also grateful for the support provided by Mia Crampin and Associate Professor Steffen Geiss, formerly of the Malawi Epidemiology and Intervention Unit (MEIRU) in Lilongwe and Karonga, who funded my accommodation and stay at Chilumba. I am sincerely grateful for the dedication and leadership of Jullita Malava and her team at MEIRU in Karonga. 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PLoS ONE 10(7):1–26. https://doi.org/10.1371/journal.pone.0133602 Wong PJ, Li MI, Chong C, Ng L, Tan C (2013) Aedes (Stegomyia) albopictus (Skuse): A Potential Vector of Zika Virus in Singapore . 7 (8), 1–5. https://doi.org/10.1371/journal.pntd.0002348 Additional Declarations The authors declare no competing interests. Supplementary Files APPENDIX.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6883143","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":470570726,"identity":"e7cf01f0-4b83-4ddd-ac08-8830b7d80e40","order_by":0,"name":"Penjani Promise Redson Chunda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYJACAwYGCRABJCuAJDNzAylazoC0MBLWAtPHIMHYBmIS0GLO3nug4OcOC3lzidyDN37OOxzN3w7U8qNiG04tlj3nEgx7z0gY7pyRl2zZu+1w7ozDjA2MPWdu43bPjRwDA942iQQgw0yCF6ilAaiFmbENj5b7bwwM/0K1SP6dczh3PkEtN3gMjGG2SPM2HM7dQEiLZU+OgbFsm4ThhjNvjK1ljqXnbgRqOYjPL+bsZ8wM37bVyRsczzG8+abGOnfe+cMHH/yowOMwBgY2AyR+M5g8gFM9RAvzAyR+HT7Fo2AUjIJRMEIBABm/WjsJG4kjAAAAAElFTkSuQmCC","orcid":"","institution":"University of Malawi","correspondingAuthor":true,"prefix":"","firstName":"Penjani","middleName":"Promise Redson","lastName":"Chunda","suffix":""}],"badges":[],"createdAt":"2025-06-12 20:58:35","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6883143/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6883143/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84666544,"identity":"3d835dc8-756b-450e-866b-402f2a4f670e","added_by":"auto","created_at":"2025-06-16 05:46:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157579,"visible":true,"origin":"","legend":"\u003cp\u003eAedes aegypti ready for a blood meal.\u003c/p\u003e\n\u003cp\u003eSource: https://www.msmosquito.org/invasive-Aedes-mosquitoes\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/49f5dc0355b08e279a730b79.jpeg"},{"id":84665564,"identity":"9b418dde-bd57-4d20-a90c-0fd823c646bb","added_by":"auto","created_at":"2025-06-16 05:38:23","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35769,"visible":true,"origin":"","legend":"\u003cp\u003eAedes albopictus mosquito\u003c/p\u003e\n\u003cp\u003eSource: INVAB Fumigation \u0026amp; Pest Ctrl Mgt Pte Ltd\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/27bfe57daa70976e308006e0.jpeg"},{"id":84664378,"identity":"1034a31b-e0e3-4ae8-9c04-978466972526","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5159479,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal map of predicted distributions of Ae. Aegypti.\u003c/p\u003e\n\u003cp\u003eSource:\u003cem\u003e \u003c/em\u003eResearchgate (uploaded by Schaffner).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/bb735553a60466e1b5fa0a9b.png"},{"id":84665563,"identity":"6f9b6d3e-41f5-4656-8a76-489c70c53871","added_by":"auto","created_at":"2025-06-16 05:38:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221715,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal map depicting the probability of Ae. albopictus occurrence Source: Wikimedia Commons\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/24fa18cb462241799b835a21.png"},{"id":84664368,"identity":"1d4058a9-8cf6-4452-b8e4-e5ec048f6f09","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":58495,"visible":true,"origin":"","legend":"\u003cp\u003eDengue and Aedes aegypti mosquitoes in Africa. Source: Researchgate (Uploaded by Fred Were, 2012)\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/01bd918f930c982c61b83bb6.jpeg"},{"id":84665565,"identity":"d1471e07-4262-48ab-8b68-2acfcbe43602","added_by":"auto","created_at":"2025-06-16 05:38:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":407915,"visible":true,"origin":"","legend":"\u003cp\u003eMap showing countries where the Chikungunya Virus has been reported. Source: CDC (https://www.cdc.gov/chikungunya/geo/index.html)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/8ed16a32999824718587f670.png"},{"id":84665566,"identity":"4e320d0b-a40c-483f-a03e-14cb411b9f9c","added_by":"auto","created_at":"2025-06-16 05:38:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":277875,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Malawi showing risk (R) based only on vector composition. Source:\u003cem\u003e D Pemba. University of Malawi Biology Department.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/5da0d8ef096db9db85bac316.png"},{"id":84664380,"identity":"cd3bd448-cc50-4482-afee-8335616ff12f","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":245625,"visible":true,"origin":"","legend":"\u003cp\u003eArbovirus life cycle. Source: southsudanjournal.com\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/9ddfa18495d861cf42286001.png"},{"id":84664385,"identity":"0fab9c32-9c52-4f90-9d47-7d3b0113c7c9","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":340016,"visible":true,"origin":"","legend":"\u003cp\u003eMap of Karonga District showing some physical features, including road infrastructure, lakes, rivers, forests, and vegetation. Source: mec.org.mw\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/59c401e053312529cf5834ec.png"},{"id":84664376,"identity":"09ce9895-22a5-4616-a21a-c8aba4dba04b","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":258697,"visible":true,"origin":"","legend":"\u003cp\u003eMosquito collection sites and the respective study villages along the north–south transect.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/9aa7604fbf6de8f08db4dc82.png"},{"id":84664374,"identity":"c3ebd74f-3820-4e7b-944b-3782aedbe4b4","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":34257,"visible":true,"origin":"","legend":"\u003cp\u003eMosquito abundance over months, cluster and rainfall amount\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/e5a48203cd50410d4c23fe42.png"},{"id":84667282,"identity":"4b14e25b-0340-47ca-8513-41f0082d9daf","added_by":"auto","created_at":"2025-06-16 05:54:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8052500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/979a6608-e424-4bfd-87e3-633a5351e312.pdf"},{"id":84664382,"identity":"99081dbd-5b5d-442d-ae0e-21fe45ff4fa1","added_by":"auto","created_at":"2025-06-16 05:30:23","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":906697,"visible":true,"origin":"","legend":"","description":"","filename":"APPENDIX.docx","url":"https://assets-eu.researchsquare.com/files/rs-6883143/v1/e84d20d86756f231ff9c9502.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eAsssessing the potential of Arboviral Transmission in Karonga District, Northern Malawi\u003c/p\u003e","fulltext":[{"header":"CHAPTER ONE BACKGROUND","content":"\u003ch2\u003e\u003cstrong\u003e1.1\u0026nbsp;Background Information\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eHistorically, Chikungunya Virus (CHIKV) was first isolated in a febrile case in the southern part of Tanzania during a dengue-like outbreak between 1952 and 1953 (WHO et al., 2016). Chikungunya Viruses belong to a genus called Old World Alpha viruses and family Togaviridae. It is an Icosahedral virus encased by lipids and is a single-stranded RNA virus\u0026ndash;60-70 nm in length with a genome of approximately 12 kilobase (kb) (Wahid et al., 2017). CHIKV is a mosquito transmitted alphavirus that is emerging as a global threat because of the highly debilitating nature of the associated disease and the unprecedented magnitude of its spread. Similar to the dengue virus (DENV), it originated in Africa and has since spread across the globe, causing large numbers of epidemics that have infected millions of people in Asia, the Indian subcontinent, Europe, the Americas, and the Pacific Islands. Similarly, dengue fever, incidence has grown in the last decade worldwide and according to the WHO, it has affected 3.9 billion people and in 128 countries are at risk of infection (Simo, Bigna, Kenmoe, et al., 2019). Approximately 390 million dengue infections occur each year in people worldwide; 96 million (25%) clinically manifest the disease, and 50% have mild disease (Bhatt et al. 2013). DENV is one of the most common arboviruses in tropical and subtropical mosquito bone viral diseases. DENV is transmitted by two main vectors, \u003cem\u003eAedes aegypti\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAedes albopictus\u003c/em\u003e. These vectors have become widely distributed across tropical and subtropical regions globally with the advent of global phenomena such as urbanization, high rate of population growth, inadequate water supply, sewers, poor waste management systems, and international travel.\u003c/p\u003e\n\u003cp\u003eAlthough originally from Africa, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e has recently been found across the world, including Mexico, Central and South America, the Caribbean, and Asia. This mosquito is popularly called the yellow fever mosquito, whereas \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e is known as the Asian tiger mosquito. \u0026nbsp;Due to their invasive nature, arboviral diseases such as dengue, zika, yellow fever, and chikungunya are increasing global public health concerns due to their rapid geographical spread in their preferred eco-epidemiological zones. Knowledge of the contemporary distribution of their shared vectors, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e, remains incomplete and is complicated by an ongoing range expansion fueled by increased global trade and travel (CDC, 2016; Kraemer et al., 2015) and climate change.\u003c/p\u003e\n\u003cp\u003eThere is a close relationship between ZIKV, DENV, CHIKV, and yellow fever virus (YFV), which currently causes a large outbreak in the Americas. Three genotypes were identified: West Africa, East Central South Africa, and Asia. Chikungunya disease is characterized by acute illness with fever \u0026gt;38.9 \u003csup\u003e0\u003c/sup\u003ec severe polyarthralgia that can last for some years, and maculopapular pruritic rash lasting for a week. Infected neonates develop serious diseases that affect the heart, skin, and brain. Bleeding and disseminated intravascular coagulation have also been observed (Goeijenbier et al., 2016; Vu \u0026amp; Labeaud, 2018; Nanev Slavov \u0026amp; Kaori Otaguiri, 2015).\u003c/p\u003e\n\u003cp\u003eIn 2010, twenty-two countries in Africa reported sporadic cases or outbreaks of dengue; 12 other African countries reported dengue only in travelers. The presence of the disease and high prevalence of antibodies to dengue virus in limited serologic surveys suggest endemic dengue virus infection in all or many parts of Africa (Amarasinghe et al., 2011)\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMalawi is one of the 13 countries in sub-Saharan Africa, which, according to Light Brown, shows that dengue has not been reported, but \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e mosquitoes, a potential vector of ZIKV, DENV, CHIKV, and YFV, unlike \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e, which has the same vectorial capacity as \u003cem\u003eAedes aegypti\u003c/em\u003e, studies have implicated \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e in transmission as the most common vector (Amarasinghe et al., 2011). Studies carried out in Singapore have demonstrated the vectorial capacity of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e to transmit ZIKV since the virus was isolated in the salivary glands, which implies the potential to transmit the virus during blood meals (Wong et al., 2013). Similarly, a study carried out by Bisimwa et al. (2016) showed that mosquito vectors of arboviruses and their associated diseases of dengue, Chikungunya, and Zika were endemic and distributed throughout the Kyela district, Tanzania, which is a district neighboring the Karonga district in the present study area with varying densities, with vectors restricted to certain areas probably due to ecological and environmental adaptation.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767773\"\u003e\u003cstrong\u003e1.2 Problem statement\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eArbovirus diseases are emerging health problems that require deliberate attention before they become a serious health problem. Factors that escalate the disease are related to vector breeding and increases in population numbers, such as increasing urbanization, poor urban planning, changes in climatic factors, and the availability of favorable micro-ecological conditions suitable for \u003cem\u003eAedes\u003c/em\u003e mosquito breeding in sub-Saharan Africa. In the face of such potential threats, there is a need for vigilance and establishment of preparedness measures before arboviral epidemics hit Malawi. Such an epidemic poses an overwhelming burden to the health system and potentially compromises the achievement of sustainable development goals (SDGs) (Baraka \u0026amp; Kweka, 2016). According to Higa et al. (2015), vector identification is crucial for controlling arboviral diseases, as the only way to reduce transmission. In Mozambique, cases were reported at the Eparse Islands in the Mozambique Channel in 2014, and several dengue cases were reported in the northern towns of Pemba, Nampula, and Mozambique. As Malawi is surrounded by countries with arboviral infection outbreaks, such as Tanzania and Mozambique, the likelihood of arbovirus transmission is very high. Since Karonga shares some characteristic geo-socio-ecological factors with neighboring countries like Tanzania and the rest of the southern region is bordered by Mozambique, the risk of transmission is very likely. In Malawi, a serological survey conducted in three districts, Blantyre (in the south), Lilongwe (in the central) and Karonga (in the northern region) established a prevalence of antibodies to these arboviruses which is indicative of previous infections. The prevalence of ZIKV antibodies was 0% in Blantyre, 0.6% (95%CI 0.0-1.7) in Lilongwe, and 2.9% (0.4-5.4) in Chilumba. The prevalence of DENV antibody was 2.2% (0.0-4.4), 5.6% (2.2-8.9) and 14.9% (9.6-20.3) at the three sites, respectively. The prevalence of CHIKV antibodies was 35% (27.6-42.4), 37.2% (30.1-44.4), and 67.2% (60.2-74.3%) at the three sites, respectively. This necessitated the need to carry out an entomological study to establish both the presence of \u003cem\u003eAedes\u003c/em\u003e mosquitoes and their importance in the transmission of arboviruses in the Karonga District.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767774\"\u003e\u003cstrong\u003e1.3 Rational/Justification\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eDespite the positive seroprevalence reported by some studies, Malawi has not yet established any surveillance system, developed any laboratory monitoring system, or any control program that deliberately targets the control of arboviruses such as ZIKV, DENV, and CHIKV. This could be because no deliberate effort has been made by the Ministry of Health (MoH) to develop control programs that aim to control these arboviruses. This may be because no large-scale studies have established the arboviral burden in the country at large. Some programs have been developed at the central level that target a few selected tropical diseases, such as malaria, \u003cem\u003eschistosomiasis\u003c/em\u003e, and \u003cem\u003elymphatic filariasis\u003c/em\u003e, among many but not arboviruses. Similarly, the National Health Management Information System (HMIS) and Integrated Disease Surveillance and Response (IDSR) do not capture or report data on arboviruses in the country. The findings of this study will establish the species range of \u003cem\u003eAedes\u003c/em\u003e mosquitoes and the potential for arbovirus transmission in the country, and hence provide scientific evidence that can inform the MoH and all relevant stakeholders to institute programs aimed at the control of arboviruses and establish a robust surveillance and control program.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767775\"\u003e\u003cstrong\u003e1.4 Hypothesis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThere are no infected arbovirus vectors present or distributed in the Karonga District in northern Malawi.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767776\"\u003e\u003cstrong\u003e1.5 Objectives of the study\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc187767777\"\u003e\u003cstrong\u003e1.5.1 Main objective\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe main objective of this study was to determine the species abundance of \u003cem\u003eAedes\u003c/em\u003e mosquitoes and their importance in arbovirus disease transmission.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc187767778\"\u003e\u003cstrong\u003e1.5.2 Specific objectives\u003c/strong\u003e\u003c/h3\u003e\n\u003col\u003e\n \u003cli\u003eTo determine the species abundance of \u003cem\u003eAedes\u003c/em\u003e mosquitoes in the Karonga district, northern Malawi.\u003c/li\u003e\n \u003cli\u003eTo assess whether the local \u003cem\u003eAedes\u003c/em\u003e mosquitoes found in the Karonga district in the northern region of Malawi carry arboviruses.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"CHAPTER TWO LITERATURE REVIEW","content":"\u003ch3\u003e\u003cstrong\u003e2.1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Arboviral mosquito vectors\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAccording to Wilkerson et al. (2015), the \u003cem\u003eAedes\u003c/em\u003e mosquitoes under study, belong to tribe \u003cem\u003eAedini\u003c/em\u003e, order Diptera and family Culicidae, genus \u003cem\u003eAedes\u003c/em\u003e, subgenus Stegomyia. The sculcllaris, albopictus, and aegypti subgroups. According to an earlier classification by Knight and Stones (1977) (Sivanathan, 2006), \u003cem\u003eAedes\u003c/em\u003e mosquitoes belong to Phylum Arthropoda; Class, Insecta; Order, Diptera; Family, Culicidae; Sub-family Culicinae; Genus, \u003cem\u003eAedes\u003c/em\u003e meaning unpleasant in Latin, also known as true flies, and it is sometimes further subdivided into subgenus Stegomyia. The \u003cem\u003eAedes\u003c/em\u003e mosquito species are \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e, from Africa and Asia, respectively.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767782\"\u003e\u003cstrong\u003e2.2 \u0026nbsp; \u0026nbsp; \u0026nbsp; Role of \u003cem\u003eAedes\u0026nbsp;\u003c/em\u003emosquitoes as arboviruses vectors\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eSimilar to most other flies in the order Diptera, Aedes mosquitoes have a life cycle that comprises eggs that hatch into adults having gone through metamorphosis to larva, pupa, and finally, adults emerge (Rozendaal, 1997). Aedes mosquitoes generally takes 7-10 days for adults to emerge from eggs, although eggs can survive for up to 8 months clinging to containers such as glue. This means that eliminating all larvae, pupae, and adult Ae. aegypti \u0026nbsp;from a site, its population could recover two weeks later as a result of egg hatching following rainfall or the addition of water to containers harboring eggs. \u003cem\u003eAedes\u003c/em\u003e mosquitoes were distinct. They differ in terms of color, preferred habitat, biting habits, vectorial capacity, and breeding location (CDC, 2012a). \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e is attracted to the chemical compounds emitted by mammals. These compounds included ammonia, carbon dioxide, lactic acid, and octenol. To date, studies done so far have shown no significant difference in terms of eggs laid in ovitraps with ordinary water or those with hay infusion and other substrates (Chadee, Lakhan, Ramdath, \u0026amp; Persad, 1993; Nazni et al., 2009). Viruses are transmitted to humans through the bites of an infective female \u003cem\u003eAedes\u003c/em\u003e mosquito, which mainly acquires the virus while feeding on the blood of an infected person. The life cycle of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e is entirely dependent on the environment created by humans. \u0026nbsp;Larvae breed from a variety of artificial containers, such as jars, discarded cans, flower vases, cement tanks, ant traps, used tires, and plastic buckets around humans. Preference for humans as hosts is an important factor for transmission. Their close association with humans contributes largely to the effective transmission of arboviruses (Brady et al., 2014; Antonio, 2009).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany studies have implicated \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e as a vector for the transmission of arboviruses, which have been proven to be competent vectors of arboviral diseases, such as dengue, chikungunya, yellow fever, and ZIKV (Calvez et al., 2016). However, it is now becoming interesting that some studies conducted recently in Africa implicate some species such as \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e (Wong et al., 2013) and found that the vector was competent for ZIKV in the laboratory in Singapore. Furthermore, Diallo et al. (2014) found that ZIKV amplification was widespread in the Ke´dougou area; in Senegal, the team involved several mosquito species as probable vectors and encompassed all investigated land cover classes except indoor locations within villages. Dengue and Chikunguya have become major public health concerns for the past 50 years due to the rapid spread and increasing disease burden worldwide. In Iran, a recent study by Bakhshi et al. (2020) showed evidence of Chikungunya virus in mosquitoes sampled from some sites under study, In the United Kingdom (UK), Chapman et al. (2020), after a study of mosquitoes that were caught in the wild and infected with arboviruses showed vectorial capacity of several arboviruses. \u0026nbsp;(Kraemer et al., 2015, Diagne et al., 2015, Powell \u0026amp; Tabachnick, 2013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the Americas, confounding results were found in a study that aimed to evaluate the vector competence of mosquitoes. \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAedes albopictus\u003c/em\u003e from the Caribbean (Martinique, Guadeloupe), North America (southern United States), and South America (Brazil, French Guiana) for the currently circulating Asian genotype of arboviruses isolated from a patient in New Caledonia in April 2014. In the Pacific region, dengue and chikungunya were seen on the rise due to the presence of the competent \u003cem\u003eAedes\u003c/em\u003e mosquito. In Iran, (Chouin-Carneiro et al. (2016) and Calvez et al. (2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn Nigeria, a study was carried out and established that not all febrile conditions present at the facility were malarial by displaying typical malaria symptoms. This is a similar observation in most of our hospitals, where most patients are characteristic of arboviral infection and not necessarily of malaria (Onyedibe et al., 2018). Another study on systematic measurement of head circumference at birth in Nigeria reported a prevalence of microcephaly (according to the WHO definition) of 10.6% in more than 3000 consecutive births in Lagos, Nigeria, in 2012 (Meda et al., 2016), establishing the presence of Zika virus. In Malawi, there are reports of Guillaine-Barre syndrome and microcephaly that are indicative of ZIKV infection among populations, and similar signs indicative of other arboviral infections.\u003c/p\u003e\n\u003cp\u003eFurther investigations are needed on the vector competence of other species associated with arboviral transmission to better understand the ecology and epidemiology of this virus. This makes the fight against arboviruses an upheaval task looking at emerging vector competencies, and needs to identify a variety of species ranges for vector competence studies.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767783\"\u003e\u003cstrong\u003e2.3 \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e (Linnae)\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe yellow fever mosquito, commonly known as \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e (Figure 1), is a mosquito that spreads dengue fever, Chikungunya, Zika, and yellow fever viruses. This mosquito can be recognized by white markings on the legs and a marking in the form of a lyre on the thorax. The mosquito originated in Africa but is now found in tropical and subtropical regions throughout the world (Sivanathan, 2006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003eis a vector for the transmission of several tropical fevers. Only female blood bites are required to mature eggs. Although \u003cem\u003eAe. aegypti\u003c/em\u003e is known to be a susceptible host for dengue, as \u003cem\u003eAedes albopictus\u003c/em\u003e and \u003cem\u003eAe, polynesiensis\u003c/em\u003e, is, it is nevertheless a better vector, because it is a highly domestic species, which breeds in containers in or around houses, and with adults resting in homes. These characteristics ensure it has a much stronger vector-human contact than the other vectors (CDC, 2012b), this is also echoed by Service, 1992,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are distinct morphological differences between male and female \u003cem\u003eAedes\u003c/em\u003e mosquitoes. Males have feathery antennae.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767784\"\u003e\u003cstrong\u003e2.4 \u003cem\u003eAedes albopictus\u0026nbsp;\u003c/em\u003e(Skuse)\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eThe tiger mosquito or forest day mosquito \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e (Stegomyia albopicta), from the mosquito (Culicidae) family, is characterized by black and white striped legs and small black and white striped bodies (Figure 2).\u003c/p\u003e\n\u003cp\u003eIt is native to the tropical and temperate regions of Asia. However, in the past couple of decades, this species has invaded many countries throughout the world through tire trade, and the global geographic distribution of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e has dramatically shifted as a result of the introduction of the species from Orient to New World, Europe, and Africa by frequent use of tires and recently due to uncontrolled urbanization in developing countries and global warming, which influence vector mosquitoes and exert an impact on vector-borne diseases. The frequent movement of people by aircraft has also resulted in the further introduction of vector mosquitoes to new places (Kraemer et al., 2015).\u003c/p\u003e\n\u003cp\u003eThis mosquito has become a significant vector in many communities because it is closely associated with humans (rather than living in wetlands), and typically flies and feeds in the daytime, in addition to at dusk and dawn. This insect is called a tiger mosquito because its striped appearance is similar to that of a tiger. \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e is an epidemiologically important vector for the transmission of many viral pathogens, including West Nile virus, yellow fever virus, St. Louis encephalitis, dengue fever and Chikungunya fever. Unlike \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e eggs have the ability to diapause (the delay in development in response to regularly and recurring periods of adverse environmental conditions) during the winter season in temperate zone Asia. Gubler suggested that the maximum egg longevity period of \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003ealbopictus\u003c/em\u003e was recorded for 243 d (Gubler, 1970).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e is a competent vector for many viruses, including dengue fever \u0026nbsp; and Eastern equine en­cephalitis virus, and has been proven to be a competent vector for ZIKV (Wong et al., 2013). Its life cycle is closely associated with the human habitat, and it breeds in containers with standing water, often tires, or other containers. As a sylvatic (forest), it is a daytime feeder and can be found in shady areas where it rests in shrubs near the ground. \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e feeding peaks in the early morning and late afternoon, and is an opportunistic and aggressive biter with a wide host range, including people and domestic and wild animals, unlike \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e, which is mostly domestic both outdoors and indoors (Rios \u0026amp; Maruniak, 2014; Brady et al., 2014; Web Image, 2012).\u003c/p\u003e\n\u003cp\u003eAe. \u003cem\u003eaegypti\u003c/em\u003e and Ae. \u003cem\u003eAlbopictus\u003c/em\u003e shares its life cycle and uses natural and artificial water-holding containers (e.g., tree holes, used tires, plastic containers, and clogged gutters) to lay eggs. After hatching, larvae grow and develop into pupae and subsequently into terrestrial, flying adult mosquitoes (CDC, 2016b).\u003c/p\u003e\n\u003ch3 id=\"_Toc187767785\"\u003e\u003cstrong\u003e2.5 Distribution of \u003cem\u003eAede\u003c/em\u003es \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eA yellow fever mosquito (\u003cem\u003eAe. aegypti\u003c/em\u003e) has a cosmo-tropical distribution and spreads to more temperate regions during the summer months. Originating in Africa, \u003cem\u003ethe Ae. aegypti\u003c/em\u003e is now present globally in the tropical and subtropical regions. Research on \u003cem\u003eAedes\u003c/em\u003e mosquitoes over the past decade has documented two morphologically distinct subspecies of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e:\u003cem\u003e\u0026nbsp;Ae\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e \u003cem\u003eformosus\u003c/em\u003e (Mattingly 1967). Knowledge of the contemporary distribution of their shared vectors, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e, remains incomplete and complicated by an ongoing range expansion fueled by increased global trade and travel. Mapping the global distribution of these vectors and the geographical determinants of their ranges are essential for public health planning. Today, about half of the world’s population is at risk of dengue infection, and chikungunya outbreaks, which were previously limited to Africa and Asia, have recently been reported in the Caribbean, South America, and Europe. Dengue and chikungunya viruses are transmitted between people by two mosquito species, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and Ae. \u003cem\u003ealbopictus\u003c/em\u003e. Therefore, it is important to identify areas at risk where these mosquito species are found around the globe. It is also important to predict where these species could become established if they were introduced to identify areas that could become at risk in the future (Kraemer et al., 2015), as shown in the maps below:\u003c/p\u003e\n\u003cp\u003eA yellow fever mosquito (\u003cem\u003eAe.\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e) has a cosmo-tropical distribution and spreads to more temperate regions during the summer months. Originating in Africa, the Ae. Aegypti is now present globally in the tropical and subtropical regions. Research on \u003cem\u003eAedes\u003c/em\u003e mosquitoes over the past decade has documented two morphologically distinct subspecies of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e: \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u003c/em\u003e \u003cem\u003eformosus\u003c/em\u003e. The two subspecies were distinguished based on the color of the tegument and abdominal scale patterns. The arrival of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e has been correlated with a decline in the abundance and distribution of the yellow fever mosquito \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e (Linnaeus). Aedes albopictus larvae outcompete Ae. aegypti larvae for food, and develop at a faster rate (\u003cem\u003eBarrera 1996 Competition and Resistance to Starvation in Larvae of Container-Inhabiting Aedes Mosquitoes Ecol Entomol 21 117-127.Pdf\u003c/em\u003e, n.d.).\u003c/p\u003e\n\u003cp\u003eFrom Figure 4, which shows the predicted distribution of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e, Malawi still falls within the region with the median probability of the vector distribution. Thus, the country established itself in terms of surveillance and response activities.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767786\"\u003e\u003cstrong\u003e2.6. \u0026nbsp; \u0026nbsp; \u0026nbsp;Arboviruses\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eArboviruses are known to affect communities globally, and some have reached the extent of outbreaks. \u003cstrong\u003e\u003cem\u003eArbovirus\u003c/em\u003e\u003c/strong\u003emeans \u003cstrong\u003ear\u003c/strong\u003ethropod-\u003cstrong\u003ebo\u003c/strong\u003erne \u003cstrong\u003evi\u003c/strong\u003eruses. The word \u003cstrong\u003earthropod\u003c/strong\u003e comes from the Greek root words \u003cem\u003earthro\u003c/em\u003e- meaning \u003cem\u003ejoint\u003c/em\u003e and -\u003cem\u003epod\u003c/em\u003e meaning \u003cem\u003efoot\u003c/em\u003e and that refers to a unique feature of the group of insect vectors. Jointed legs, also called appendages, vary widely in number and function. Appendages were used for eating, feeling, sensing, mating, respiration, walking, and defense. Arboviruses are categorized into five families: Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, and Rhabdoviridae. Arboviruses vary in their size and shape. Most are spherical, except for Rhabdoviridae, which is bullet-shaped. Spherical viruses exhibit icosahedral symmetry. Virions range in size from approximately 45 nm in diameter (flaviviruses) to \u0026gt; 380 nm in length (some rhabdoviruses). The genomes of almost all the arboviruses contain RNA. RNA may be single- or double-stranded, linear or circular, and may be positive or negative. Positive-sense RNA can act as mRNA, and genomes with this type of RNA are termed infectious. On the other hand, genomes with negative-sense RNA must first make positive-sense RNA for transcription and are termed noninfectious (Simpson, 1972). These arboviruses are notable for causing serious diseases such as dengue, Zika, and Chikungunya, which sometimes occur during an outbreak and cause mortality in society. This study focused mainly on dengue and chikungunya. Chikungunya virus belongs to the genus Alphaviruses and family Togaviridae, which are of medical importance because they have caused major outbreaks worldwide. This study focuses mainly on dengue and Chikungunya\u003c/p\u003e\n\u003ch3 id=\"_Toc187767787\"\u003e\u003cstrong\u003e2.6.1 \u0026nbsp; \u0026nbsp;Dengue virus\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eDengue virus falls under the Genus Flavirus and family Flaviviridae, similar to Zika Virus. The presence of the vector and the diagnosed disease in some countries has encouraged us to develop systems for entomological surveillance and response. Most parts of Africa have recorded the presence of \u003cem\u003ethe Aedes\u003c/em\u003e mosquito, although others have not yet. A systematic review by Simo et al. (2019) noted that although a total of 22 countries in Africa reported sporadic cases of outbreaks of dengue fever, to date, no study has accurately investigated the epidemiology of DENV infection among febrile and apparently healthy populations in this continent and thus need to do so.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5, dengue was reported among travelers in 34 countries in which dengue has been reported, including dengue reported only in travelers and \u003cem\u003eAe\u003c/em\u003e. \u003cem\u003eaegypti\u0026nbsp;\u003c/em\u003emosquitoes as shown by the brown colour indices. Light brown indicates 13 countries (Mauritania, The Gambia, Guinea-Bissau, Guinea, Sierra Leone, Liberia, Niger, Chad, Central African Republic, Republic of the Congo, Malawi, Zimbabwe, and Botswana) in which dengue has not been reported but that have \u003cem\u003eAe. aegypti\u0026nbsp;\u003c/em\u003emosquitoes. White indicates five countries (Western Sahara, Morocco, Algeria, Tunisia, and Libya) for which data on dengue and \u003cem\u003eAe. aegypti\u0026nbsp;\u003c/em\u003emosquitoes were unavailable (Were 2012).\u003c/p\u003e\n\u003ch3 id=\"_Toc187767788\"\u003e\u003cstrong\u003e2.6.2 \u0026nbsp; \u0026nbsp;Chikungunya Virus\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eUnlike DENV, CHIKV is widely isolated in Africa and the rest of the world (Figure 6). Contemporaneous systematic meta-analysis prevalence study by F. B.N. Simo et al., 2019, established a high prevalence of CHIKV infection in \u0026nbsp;Africa, especially during outbreak periods. Chikungunya disease has been identified in nearly 40 countries, including half of them in Africa. Although endemic to Asia and Africa, the virus has invaded new territories, including the Indian Ocean island and Italy. This has made it a public health concern, and hence the need for awareness and response (CDC \u0026amp; PAHO, 2011).\u003c/p\u003e\n\u003cp\u003eBased on the chronology of the Chikungunya outbreak, Wahid et al. (2017) In Africa, CHIKV was first reported in Tanzania in 1952. This was followed by several other epidemics in the Central African Republic, Guinea, Burundi, Angola, Uganda, Malawi, Nigeria, Democratic Republic of the Congo, and several other states.\u003c/p\u003e\n\u003cp\u003eFrom the 1960s to the 1990s, outbreaks were recorded in the Democratic Republic of the Congo, Central African Republic, Malawi, Uganda, Burundi, Angola, Guinea, South Africa, and Nigeria. Almost half of the cases were reported in June 2004 during \u0026nbsp;an \u0026nbsp; outbreak that occurred in Lamu Atoll, Kenya. The first outbreak in Malawi occurred between 1987–89. A few cases have been reported in 2001 and 2015. \u0026nbsp;As of October 30, 2020, Malawi has been in countries where Chikungunya has been reported (CDC, 2016a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMalawi falls in countries where chikungunya has been reported; however, few studies have been conducted to ascertain the extent of the outbreak.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Biology Department of Chancellor College carried out a Risk Map for various vectors and their associated diseases being transmitted. Karonga was marked as an area at high risk of arboviruses (Figure 7). The map showed that Karonga and other districts were at the highest risk of arboviruses.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eR\u0026nbsp;\u003c/strong\u003earbovir=\u003cstrong\u003ep\u003c/strong\u003e (imm+dens) + \u003cstrong\u003e\u0026beta;\u003c/strong\u003e (env temp/alti+precip) + \u003cstrong\u003e\u0026alpha; (\u003c/strong\u003evecden+ type+\u003cstrong\u003ec\u003c/strong\u003e........\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ch3 id=\"_Toc187767789\"\u003e\u003cstrong\u003e2.7 \u0026nbsp; \u0026nbsp; \u0026nbsp; Life cycle of arboviruses\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eArboviruses are maintained in nature through biological transmission between susceptible vertebrate hosts by blood-feeding arthropods (mosquitoes, psychodids, ceratopogonids, and ticks). Vertebrate infection occurs when the infected arthropod takes a blood meal. The term 'arbovirus' has no taxonomic significance (States, 2007). Arboviruses have several types of life cycles, but many have a sylvatic cycle, such as \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e, while some also have an urban cycle, \u0026nbsp;the sylvatic cycle (sometimes known as the jungle cycle). The viral cycles between an arthropod and a mammalian host with humans are usually a dead-end host infected by the arthropod and urban cycle. The virus cycles between humans and an arthropod species.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is an urban cycle for yellow fever and dengue fever (both also have a sylvatic/jungle cycle). \u0026nbsp;If there is an urban cycle, using window screens, bed nets, etc., to prevent access of mosquitoes to viremic patients may reduce transmission (Ayres, 2016).\u003c/p\u003e\n\u003cp\u003eArboviruses are mainly transmitted by \u003cem\u003eAedes\u003c/em\u003e mosquitoes and are widespread in both urban and peri-urban areas. Zoonotic relationships are known to occur between humans and non-human primates. The role of other reservoirs in sexual transmission remains unclear.\u003c/p\u003e"},{"header":"CHAPTER THREE METHODOLOGY","content":"\u003ch2\u003e\u003cstrong\u003e3.1 Study Area\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe study was carried out in the Karonga district, which is located in northern Malawi between the latitudes and longitudes of -10.362124 and 34.206370, respectively. Karonga District is bordered by Chitipa District to the West and Rumphi District to the South (Figure 10). The district headquarters is located approximately 50 km south of the Tanzanian border and 585 km north of the capital city of Malawi, Lilongwe. The total land area of the district is 3355 square kilometers making for 3.5% of the total land area of Malawi (94276 sq km).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.2 Topography and Climate\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(Council, 2011)\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;The relief of the district has a wide range of 400m at the lakeshore and 2400m at the high Nyika Plateau. It appears predominantly hilly, going westward from the lake. The most outstanding landforms can be divided into three zones: a) High Hills and Plateau Zone, b) Rift Valley Escarpment Zone, and c) Lakeshore Plain Zone. Similar to other districts in the country, Karonga has a subtropical climate.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003e3.3 Study\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDesign\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis is a cross-sectional study. \u0026nbsp;Sampling followed a transect design from south to north of the district, with six sampling points established. Sampling was carried out once a week over a period of three months. The location of the study was selected using existing health clusters, viz: Hara, Karonga Boma, Kayelekera, Iponga, and Kaporo, and simple random sampling was performed where all areas under the cluster had an equal chance of being selected. Simple random selection was performed for all villages under the cluster to determine the placement of traps in villages from the clusters that met the criteria for the possible habitation of \u003cem\u003eAedes\u003c/em\u003e mosquitoes. A single location was determined, and traps were placed at the sampled locations in each cluster (Figure 10).\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767795\"\u003e\u003cstrong\u003e3.4 Weather data\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eWeather data were obtained from the Metrological Department during the study period from January 2018 to December 2018.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp id=\"_Toc187757368\"\u003eTable\u0026nbsp;1: Weather data for Karonga District\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"443\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeather data for Karonga district – 2018\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eYear\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eMonth\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eRainfall\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eMax_Temp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eMin_Temp\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eWindspeed\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eJan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e180.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e30.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e21.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eFeb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e124.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e21.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e219.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e30.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eApr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e234.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e29.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eMay\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e29.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e19.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eJun\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eJul\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eAug\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e29.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eSep\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e19.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eOct\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e32.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e21.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNov\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e33.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e22.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eDec\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e198.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e31.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSource: Metrological department. Blantyre, Malawi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767796\"\u003e\u003cstrong\u003e3.5 \u0026nbsp; \u0026nbsp; \u0026nbsp; Mosquito trapping and sampling\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc187767797\"\u003e3.5.1 Sampling of mosquitoes catching sites\u003c/h3\u003e\n\u003cp\u003eIn this study, sampling targeted all life cycle stages of \u003cem\u003eAedes\u003c/em\u003e mosquitoes (larvae, pupae, and adults) present in the selected communities.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc187767798\"\u003e3.5.2 \u0026nbsp; \u0026nbsp;Mosquito trapping\u003c/h3\u003e\n\u003cp\u003eOvitraps were distributed in the selected transect points in the urban (Karonga town at Mwangwambila village, and Kaporo at Mwenengolongo and Kasebe) and rural ( Hara at Hangalawe, Iponga at Weleweta and Kayelekera at Sere village) areas of Karonga district (Figure 10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOvitraps were deployed for one week, and on the seventh day, adult and aquatic mosquito stages were sampled from traps. This routine was followed to avoid production of the second-generation mosquito family.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOnce the adult mosquitoes laid eggs in the traps, they were trapped inside, and both emerged adults (F\u003csub\u003e1\u003c/sub\u003e) and first-generation adults (F\u003csub\u003e0\u003c/sub\u003e) were collected. In addition, mosquito larvae were collected using dippers from artificial breeding habitats such as discarded tyres, troughs, holes, and other containers. Larval samples were placed in small containers and placed in rearing cages until adults emerged. After emergence, the adult mosquitoes were collected using a pen siphon, and each was placed in its own Eppendorf tube labeled according to site and placed in a-80\u003csup\u003e0\u003c/sup\u003eC refrigerator. Mosquitoes were transported using liquid nitrogen from Karonga to the laboratory at Chancellor College (now the University of Malawi). Using a dissecting microscope, \u003cem\u003eAedes\u003c/em\u003e mosquitoes were morphologically identified using a mosquito key (BUXTON, 1941) and separated from other types of mosquitoes (Kean et al., 2015). Because arboviruses are vertically transmitted, all \u003cem\u003eAedes\u003c/em\u003e mosquitoes were preserved in 90% ethanol and stored at -80°C for further analysis.\u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc187767799\"\u003e\u003cstrong\u003e3.5.6 \u0026nbsp; \u0026nbsp;Mosquito Data Collection\u003c/strong\u003e\u003c/h3\u003e\n\u003ch3 id=\"_Toc187767800\"\u003eMosquito data\u003c/h3\u003e\n\u003cp\u003eMosquito ovitraps and larval rearing provided tangible mosquito counts at each study site.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp id=\"_Toc187757369\"\u003eTable\u0026nbsp;2: Showing Mosquito collection sites and the number of ovitraps deployed for Karonga District\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"463\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealth cluster\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eShort code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVillage name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of ovitraps\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHara\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2HA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHangalawe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eIponga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4IP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGweleweta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaporo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5PK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMwenengolondo and Kasebe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaronga Town\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1KA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMwangwambila\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKayelekela\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3KY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSere\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2 id=\"_Toc187767801\"\u003e\u003cstrong\u003e3.6 \u0026nbsp; \u0026nbsp; \u0026nbsp; Mosquito processing and analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003ch3 id=\"_Toc187767802\"\u003e3.6.1 \u0026nbsp; \u0026nbsp;Isolation of RNA\u003c/h3\u003e\n\u003cp\u003eA Qiagen RNA extraction kit was used for RNA processing (Santos et al., 2004). Twenty mosquitoes from each site were pooled into 2 ml a single centrifuge tube as pooling method for all sites (Table 2). For each site, 500 µL of medium was added, ground using a pestle with a pestle homogenizer, and manually homogenized. The medium was centrifuged at 6,000xg (8,000 rpm) for 10 min. A total of 140 µL of the specimen was removed for RNA extraction and the remaining mixture was transferred to a screw-cup tube. The screw cup tube was labeled and the pestle was washed by rinsing in Jik. The stored homogenate RNA carrier was in a-80\u003csup\u003e0\u003c/sup\u003eC freezer. Homogenate carrier RNA was stored and subjected to RNA extraction, which included lysis, washing, elution, and extraction of pure RNA. Carrier RNA was added to buffer AVL. A buffer (310 µL of AVE buffer was added to a tube containing the lyophilized carrier RNA. Mixing was performed by inverting the tube ten times to reconstitute the carrier RNA. A total of 310 µL of dissolved carrier RNA was transferred to the buffer AVL. For six samples, the net was 33.6 µl which was stored in a refrigerator. A total of 560 µL of buffer AVL with carrier RNA was pipetted into a 1.5 ml centrifuge tube. A total of 140 µL of specimen was added to the mixture, pulse vortexed for 15 s, and then stored for 10 min at room temperature. The mixture was briefly centrifuged for 10 s, 560 µL of ethanol was added to the sample, vortexed for 15 s, and briefly centrifuged. The solution mixture (630 µL) was added to a QIAamp spin column (2 ml without wetting the lid and closing the cap. The mixture was centrifuged at 6,000 x g and 8,000 rpm for 1 min. The QIAamp spin column was placed into a clean 2 ml collection tube, and the tube containing the filtrate was discarded. The preceding process was repeated, the QIAamp spin column was carefully opened, and 500 µL of buffer AWI was added. The mixture was centrifuged at 8,000 rpm for 1 min. The QIAamp spin column was placed in a clean 2 ml collection tube, and the collection tube containing the filtrate was discharged. The QIAamp was carefully opened, and 500 µL of buffer AW2 was added, cap closed, and centrifuged at 13,500 rpm for 3 min. QIAamp was placed in new 2 ml centrifuge tubes, and the old filtrate was discarded. Sixty microliters of AVE buffer were added to the center of the column, and the cap was closed. The mixture was incubated at room temperature for 1 min and then centrifuged at 8,000 rpm for 1 min. The final eluate contained 90% of RNA. Pure RNA was stored in a 2 ml centrifuge tube at -80\u003csup\u003e0\u003c/sup\u003eC freeze.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767803\"\u003e3.6.2 \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction- Reverse Transcription (RT-PCR)\u003c/h3\u003e\n\u003cp\u003eThis converts single-stranded RNA into complementary DNA (cDNA) for PCR. This reaction involved reverse transcription, denaturation, annealing, and extension. The RT-PCR reagents were put into one master mix tube totaling to 95µl and centrifuged. This comprised 4µl 5X RT buffer, 2µl dNTP, 0.5µl cFD2 as forward primer, 1µl RT enzyme polymerase, 11.5µl PCR water, 1µl template RNA. For five sites, the total was 95µl and each site was 20µl. The thermocycler was programmed in three steps as follows:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eOne cycle for 42\u003csup\u003e0\u003c/sup\u003eC for 20 minutes, then\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;99\u003csup\u003e0\u003c/sup\u003eC for 5 min and\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;4\u003csup\u003e0\u003c/sup\u003eC for ∞\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe processes above complete the reverse transcription and was done for both dengue and Chikungunya. The final product produced was cDNA (Domingo \u0026amp; Patel, 2012, Petz et al., 2014) \u0026nbsp;\u003c/p\u003e\n\u003ch3 id=\"_Toc187767804\"\u003e3.6.3 \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction (PCR)\u003c/h3\u003e\n\u003cp\u003eThe master mix for the PCR assay was prepared totaling volume of 97.5µl. Extracted pure RNA was added to each tube for analysis. There were five tubes each representing a sentinel site for mosquito sample traps or rearing. For dengue and Chikungunya, the mix was as follows: 4µl 5X RT buffer, 1µl dNTP, 0.5µl dengue 802 as forward primer for dengue and 0.5µl cFD2+MAMD as reverse primer for dengue; 0.5µl CHIKVFLY as forward primer for CHIKVFLY and 0.5µl cFD2+MAMD as reverse primer for CHIKVFLY; 0.5µl CHIKV E1 as forward primer for CHIKV E1 and 0.5µl cFD2+MAMD as reverse primer for CHIKV E1, 0.25µl polymerase, 12.75µl PCR water, 0.5µl template RNA. The reaction tube was 20µl. This was performed for each primer for dengue, chikungunya, CHIKV FLY, and CHIKV E1 (Scaramozzino et al., 2001; Lee et al., 2012).\u003c/p\u003e\n\u003cp\u003eDistributed the 19.5µl master mix was distributed into each tube for the five sites using short codes denoted at each site. The tubes were capped and vortexed to mix reagents.\u003c/p\u003e\n\u003cp\u003eThe thermocycler run for each was programmed as follows:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFor CHIKV E1 and FLY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe steps are as follows.\u003c/p\u003e\n\u003cp\u003e94\u003csup\u003e0\u003c/sup\u003eC for 3 min for one cycle. Then for 30 cycles at:\u003c/p\u003e\n\u003cp\u003e98\u003csup\u003e0\u003c/sup\u003eC for 20 sec \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e57\u003csup\u003e0\u003c/sup\u003eC for 20 sec \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e72\u003csup\u003e0\u003c/sup\u003eC for 40 sec \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4\u003csup\u003e0\u003c/sup\u003eC for ∞\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFor dengue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe steps are as follows.\u003c/p\u003e\n\u003cp\u003e94\u003csup\u003e0\u003c/sup\u003eC for 3 min for one cycle. Then for 30 cycles at:\u003c/p\u003e\n\u003cp\u003e98\u003csup\u003e0\u003c/sup\u003eC for 20 sec\u003c/p\u003e\n\u003cp\u003e60\u003csup\u003e0\u003c/sup\u003eC for 20 sec \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e72\u003csup\u003e0\u003c/sup\u003eC for 1 min \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4\u003csup\u003e0\u003c/sup\u003eC for ∞\u003c/p\u003e\n\u003cp\u003eThe PCR tubes were loaded in a thermocycler, the lid was closed, and the PCR program was run as indicated for each arbovirus, as described above.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767805\"\u003e3.6.4 \u0026nbsp; \u0026nbsp;Gel electrophoresis\u003c/h3\u003e\n\u003cp\u003eIt is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. The gel electrophoresis apparatus consists of a gel, often made from agar or polyacrylamide, and an electrophoretic chamber (typically a hard plastic box or tank) with a cathode (negative terminal) at one end and an anode (positive terminal) at the opposite end. An electric current was applied to move the molecules to be separated through a gel. Pores in the gel work like sieves, allowing smaller molecules to move faster than larger molecules.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, DNA was separated using agarose gel electrophoresis, where the DNA was loaded into pre-cast wells made of a gel comb, and an electric current was applied through the apparatus. The phosphate backbone of the DNA (and RNA) molecule is negatively charged; therefore, when placed in an electric field, the DNA fragments migrate to the positively charged anode. Since DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe gel was prepared using a 2% agarose gel with 1X TAE buffer as an electrolyte in a charged electric field.\u0026nbsp;For 2% agarose gel preparation, we measured 2 g of agarose in an Erlenmeyer flask containing 75 ml 1x TBE buffer. Agarose (1.5 g) was weighed, put in 1X TAE buffer, and mixed. The flask was covered with Kimwipes and heated in a microwave until the agarose was dissolved for two minutes. It was left to cool to approximately 60°C on the bench for several minutes, so the agarose should not solidify. The agarose solution was stained with 5 µL GelGreen Nucleic Acid 10000 × in water. The mixture was prepared by swirling a flask. Agarose was poured into the mold (75 ml and 25 ml at room temperature to a gel thickness of 3-5 mm. The comb was placed on top of the trays and was carefully removed after 30 min at room temperature. The gel was then positioned in an electrophoresis tank. A 1X buffer was added as an electrolyte to cover the gel to a depth of approximately 5 mm.\u003c/p\u003e\n\u003cp\u003eThe wells of the gel were loaded with a mixture of 8µl\u0026nbsp;of DNA samples or PCR products and with loading dye 2µl\u0026nbsp;bromophenol blue in each tube to a total mixture of 10µl\u0026nbsp;using pipettes slowly. Each slot represents a sample from a designated capture site. Gel wells were filled with GeneLadder 100, negative control (PCR water), negative control with polymerase, positive control with CHIKV E1, Samples 1KA, 2HA, 3KY, 4IP and 5KP representing catching sites, then well with samples for the sites for CHIKV Fly. For Chikungunya, both samples were run on the same gel. The dengue gel was run separately.\u003c/p\u003e\n\u003cp\u003eElectrical leads were switched on, and DNA in the gel moved towards the anode (red lead) after the application of a voltage of 1-5V/cm. The gel was run until the gel-loading buffer stain migrated to the appropriate distance (normally, until the bromophenol blue dye front migrated ¾ of the way down the gel). The current was turned off and the leads were removed. The gel was examined for dengue and chikungunya separately, and the observed bands were observed in the ultraviolet transilluminator gel green and photographed, as shown in the results section in this write-up (Lee et al., 2012).\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767806\"\u003e\u003cstrong\u003e3.7 \u0026nbsp; \u0026nbsp; \u0026nbsp; Data management and analysis\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eData were collected using tablets; each data point was coded, and coordinates were obtained using a global positioning system [GPS]. Data were collected using the Open Data Kit (ODK), which was installed in tablets for the collection of vital data elements needed for analysis. Data were analyzed using Microsoft Excel to generate descriptive statistics. for the weather and mosquito data per site, respectively. The results are presented as graphs and tables, with the corresponding proportions.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767807\"\u003e\u003cstrong\u003e3.8 \u0026nbsp; \u0026nbsp; \u0026nbsp; Ethical considerations\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe study did not involve household surveys or interviews; hence, sample collection did not interfere with human activity. This study was a continuation of the human study for arboviral serological study in people who already had ethical approval through the College of Medicine and Malaria Alert Centre.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767808\"\u003e\u003cstrong\u003e3.9 \u0026nbsp; \u0026nbsp; \u0026nbsp; Study limitations\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe study analyzed all potential arboviruses likely to be present in the study region, including Zika, but the availability of reagents was a limiting factor. Only DENV and CHIKV were investigated using the available primers. Lengthy procurement processes are challenging. Molecular reagents are not internally sourced; therefore, it takes much longer to procure them. Consequently, the samples took much longer to analyze and, in the process, some stored samples degraded, resulting in non-amplification (null scoring). Another factor could be poor coordination and communication between the collaborators as the collaborators involved Malaria Alert Centre, MEIRU, and Chancellor College.\u003c/p\u003e"},{"header":"CHAPTER FOUR RESULTS AND DISCUSSION","content":"\u003ch2\u003e\u003cstrong\u003e4.1 \u0026nbsp; \u0026nbsp; \u0026nbsp; Types of mosquitoes in Karonga District\u003c/strong\u003e\u003c/h2\u003e\n\u003ch2 id=\"_Toc187767812\"\u003e\u003cstrong\u003e4.1.1 \u0026nbsp; \u0026nbsp;Mosquito species collected in Karonga District\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eA total of 1,100 mosquitoes were collected across the six study sites, with 578 (53%) belonging to the other culicines and 522 (47%) \u003cem\u003eAedes aegypti\u0026nbsp;\u003c/em\u003e(Table 3). No \u003cem\u003eAe. albopictus\u003c/em\u003e (0.0%) was detected across the sampling sites. Of the total mosquitoes collected, 249 (23%) were originally collected as larvae from discarded tyres or troughs, compared to 861 (77%) adults collected using ovitraps. Ovitraps were more successful at sampling \u003cem\u003eAedes\u0026nbsp;\u003c/em\u003emosquitoes than larval sampling. However, only two places had both methods of collection, viz, Kaporo and Karonga, yielding 49% and 51% of \u003cem\u003eAedes\u003c/em\u003e mosquitoes through tire collection. Table 3 shows the mosquito yield per site and their corresponding proportions.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc187757370\"\u003eTable\u0026nbsp;3: A summary of Aedes spp, culicines, and their proportions recorded per site\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"594\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAedes albopictus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eOther culicines\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHara\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e38.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eIponga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e27.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaporo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e31.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaronga Town\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKayelekera\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e107\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e522\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e578\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2 id=\"_Toc187767813\"\u003e\u003cstrong\u003e4.1.2 \u0026nbsp; \u0026nbsp;Aedes mosquito transect distribution across Karonga District\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eKaporo yielded the majority of \u003cem\u003eAe. aegypti\u003c/em\u003e mosquitoes (31.6%), followed by Iponga (27.6%), Karonga Town (24.5%), Hara (15.7%), and Kayelekera (0.6%) had the least mosquitoes. This means that the population densities of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e mosquitoes decreased from the farther north as you went to the southern part of the district (Figure 10).\u003c/p\u003e\n\u003ch2 id=\"_Toc187767814\"\u003e\u003cstrong\u003e4.1.3. \u0026nbsp; Distribution of Aedes mosquito densities per month\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eIn the first month of March, only one \u003cem\u003eAe. aegypti\u003c/em\u003e mosquito, representing 0.19% (n=522) from Hara, making it the month with least mosquito yield of mosquitoes. The highest Aedes mosquito yield was recorded in April (81.23%), followed by May (18.58%). Kaporo registered the highest yield (32%) at all sites (Figure 11).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe rainfall season was from December to May. Heavy rainfall was recorded in April (234.5 millimeters rainfall, followed by March receiving 219.1 millimeters with May (0 mm). During the peak month of the rainy season in April, all sites recorded high mosquito densities, except for Hara. However, Hara recoded the highest number of mosquito densities in May where there was no rainfall recorded than any other site (45/97) representing 46%, followed by Iponga (41/97) representing 42%, while Kaporo and Kayerekera had zero mosquito respectively in the same month. Kaporo, which had the highest percentage of \u003cem\u003eAedes aegypti\u003c/em\u003e mosquito (32%) than any other site, recorded such a yield in April, which also recorded the highest rainfall in the three months of the study. \u0026nbsp;There was a sharp decline in mosquito densities at all sites in May, which did not record any rainfall. Kayelekera recorded only three mosquitoes during the study period, which was also recorded in May.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767815\"\u003e\u003cstrong\u003e4.2 \u0026nbsp; \u0026nbsp; \u0026nbsp; Presence of arboviruses in the vector Aedes aegypti mosquitoes\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe final results of the laboratory processes described in sections 3.4.3, 3.4.4, and 3.4.5, which highlight the laboratory analysis of RNA isolation, reverse transcription PCR, PCR, and viewing in the ultraviolet transilluminator Gelgreen separately of the \u003cem\u003eAedes aegypti\u0026nbsp;\u003c/em\u003emosquito sample, showed the presence of both Chikungunya and dengue arboviruses as reagents used to detect these viruses. All mosquito catching sites had mosquitoes that were infected with these arboviruses (Table 4).\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc187757371\"\u003eTable\u0026nbsp;4: Presence of arboviruses in mosquitoes per site across Karonga district in northern Malawi\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"594\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eHealth cluster\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSite name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of ovitraps\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNumber of \u003cem\u003eAedes\u003c/em\u003e mosquitoes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePresence of arboviruses (CHIKV and DENV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eHara\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHangalawe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eIponga\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGweleweta\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e144\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaporo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMwenengolondo and Kasebe\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKaronga Town\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMwangwambila\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eKayelekela\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSere\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003e54\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e522\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eEach site showed visible bands after electrophoresis for both the chikungunya virus and dengue virus.\u0026nbsp;\u003c/p\u003e\n\u003ch1 id=\"_Toc187767816\"\u003e\u003cstrong\u003e4.3 Discussion\u003c/strong\u003e\u003c/h1\u003e\n\u003ch2 id=\"_Toc187767817\"\u003e\u003cstrong\u003e4.3.1 Types of Mosquitoes in Karonga District\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e was present in all traps throughout the district, indicating that it was widespread. These vectors has been proven to be globally distributed (Diagne et al., 2015, Kraemer et al., 2015, Simard et al., 2005, Farajollahi \u0026amp; Nelder, 2009). The study observed that \u003cem\u003eAedes\u003c/em\u003e mosquitoes were inversely distributed to other culicines (\u003cem\u003eCulex sp.\u003c/em\u003e and \u003cem\u003eMansonia sp.\u003c/em\u003e). \u0026nbsp;A \u0026nbsp; nationwide study carried out from November 2011 to April 2012 by Maekawa et al. (2021) established the presence of these \u003cem\u003eCulex\u003c/em\u003e complexes across the country, and the species was confirmed to be spread across Africa according to some studies. Interestingly, the urban town of Karonga recorded almost equal numbers of known vectors, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e, and other culicines (50%). Kayerekera Mine yielded the highest percentage (97%) of culicines (107) compared to the known vector \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u0026nbsp;\u003c/em\u003e(3%) and had the least number of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e than all study areas. This is in contrast with another entomological study carried out to determine risk factors for anopheline in rural and urban areas in Blantyre by Mzilahowa et al. (2016), who found that rural areas had a higher risk than urban areas. However, migration factors have been attributed to malaria-related morbidity due to malaria in urban areas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStudy sites that yielded the highest numbers of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e mosquitoes, such as Kaporo and Iponga, yielded the lowest numbers of other culicines, 12.6% and 8.3%, respectively, compared with sites that yielded fewer \u003cem\u003eAedes aegypti\u003c/em\u003e, such as Hara, Kayelekera, and Karonga, which yielded 38.4%, 18.5%, and 12.6%, respectively, indicating differences in the presence of breeding habitats. The presence of suitable breeding habitats might partly explain the distribution of \u003cem\u003eAedes aegypti\u003c/em\u003e and Culicines along the sampling sites. Similar studies conducted in Kinondoni district and northern Tanzania in Kilimanjaro by Hertz et al. (2016) and Ngingo et al. (2022) found similar results, with only \u003cem\u003eAedes aegypti\u003c/em\u003e found in the study area. On the contrary, studies conducted in Senegal (Diagne et al., 2015) have shown that \u003cem\u003ethe Aedes\u003c/em\u003e \u003cem\u003ealbopictus\u003c/em\u003e vector is a competent vector to transmit both dengue and Chikungunya, even though \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e stands out as the best competent vector. In Mozambique, a survey in 32 urban areas similarly found that only 0.03% of the total samples collected from 2,807 were \u003cem\u003eAedes albopictus\u003c/em\u003e, thus corroborating the findings of this study, which did not find \u003cem\u003eAedes albopictus\u0026nbsp;\u003c/em\u003e(Ab\u0026iacute;lio et al., 2018). Information from the vector control unit at the Kayelekera mine site indicated that the area underwent vector control to stop mosquito breeding, showing that mosquito control is effective against \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e and not against other culicines that are prevalent in the area.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767818\"\u003e\u003cstrong\u003e4.3.2 Distribution of Aedes Mosquitoes in Karonga District\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eMweya et al., 2013 and Bisimwa et al., 2016 established presence of both mosquito vector densities as well as presence of arboviruses in the vectors in their study in Mbeya and Kyera districts in Tanzania which lies to the north of Karonga district, Malawi. Kaporo and Iponga, which are in the northern part, share almost the same geographical and climatic features as the neighboring districts in Tanzania; in this study, these sites registered more mosquitoes than other sites in the south. This may indicate a trend of mosquitoes in motion, as \u003cem\u003eAedes\u003c/em\u003e mosquitoes can easily be transported in moving objects, containers, and even cars and pose a risk of spreading to non-habitable areas. In Spain, the Peruvian Amazon, and Ethiopia, different studies carried out by different researchers confirmed the dispersion of \u003cem\u003eAedes albopictus\u003c/em\u003e and \u003cem\u003eAedes aegypti\u0026nbsp;\u003c/em\u003eand confirmed that vehicles such as cars and boats in addition to containers were responsible for the dispersion of these arboviral vector mosquitoes (Eritja et al., 2017, Getachew et al., 2015; Guagliardo et al., 2015). This is proof of vector dynamics due to many geosocial and anthropometric factors, and could be one of the possible factors of \u003cem\u003eAedes aegypti\u003c/em\u003e spread throughout the district. The presence of \u003cem\u003eAedes\u003c/em\u003e mosquitoes alone ascertains the potential for arboviral transmission according to Weetman et al. (2018), who found that \u003cem\u003eAedes\u003c/em\u003e mosquitoes were implicated in arbovirus transmission more than other species. However, in Iran, Bakhshi et al. (2020) found evidence of some pooled 6 samples from non-Aedes\u003cem\u003e\u0026nbsp;sp.\u003c/em\u003e, \u003cem\u003ewhich were\u003c/em\u003e positive for the Chikungunya Asian type. This however, does not mean that some of these vectors have the competency to transmit the arboviruses as efficient as \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e which is a known competent vector or arboviruses (Bisimwa et al., 2016; Chapman et al., 2020 \u0026nbsp;and Calvez et al. 2016). Similar to Tanzania and Mozambique, which are neighboring countries, \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e was found in large numbers and caused outbreaks in the respective areas and throughout the country (Higa et al., 2015, Mweya et al., 2013). \u0026nbsp; This could be true for our country since the presence of \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e throughout the district of Karonga would seriously put people at risk, as Karonga borders Tanzania and Zambia.\u003c/p\u003e\n\u003cp\u003eSuspected outbreaks of chikungunya and dengue outbreaks in Zambezia Province necessitated a nationwide survey as people were presenting at facilities with arboviral infections. According to the findings of the study by Mugabe et al., 2018, the patients were positive to Chikungunya and dengue and the abundance of the \u003cem\u003eAedes aegypti\u003c/em\u003e throughout Mozambique, entails a great risk to Malawi as the majority of Malawi\u0026rsquo;s southern part is bordering Mozambique. An unpublished report by the Biology Department at the University of Malawi, Chancellor College by Pemba et al.. established a risk map based on vector composition, and Karonga was marked as a high-risk area for arboviral transmission (Figure 7). Therefore, Malawi should be vigilant and should institute a nationwide survey to determine the presence of arboviruses in mosquitoes.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767819\"\u003e\u003cstrong\u003e4.3.3 Seasonal distribution of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAedes mosquito in Karonga district, northern Malawi\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eSeasonal changes and temperature had a direct impact on the abundance of \u003cem\u003eAedes aegypti.\u003c/em\u003e This study found more \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u0026nbsp;\u003c/em\u003eduring the month of April and towards the end of the rain, with March and April recording 219.1 mm and 234.5 mm respectively. The same month of April recorded 29.5\u003csup\u003e0\u003c/sup\u003eC and March 30.2\u003csup\u003e0\u003c/sup\u003eC and thus the range was favourable for \u003cem\u003eAedes\u0026nbsp;\u003c/em\u003emosquitoes and provided optimal breeding. Akram et al., 2009, in his study on seasonal distribution and species of daytime biting mosquitoes found that \u003cem\u003eAedes\u003c/em\u003e mosquitoes population density rapidly increased by 26.3% following the rainy season in July with temperatures between 38\u003csup\u003e0\u003c/sup\u003eC-42\u003csup\u003e0\u003c/sup\u003eC. However, in the same study, he found a population density decline in mosquitoes when the temperature reached approximately 45 to 50\u003csup\u003e0\u003c/sup\u003eC. Our results are consistent with findings from Yaound\u0026eacute; Cameroon, Djiappi-tchamen et al. (2021), where the abundance and distribution of Aedes mosquito species in each ecological setting was significantly different between the dry and rainy seasons (p \u0026lt; 0.0001). A higher density of Aedes mosquito species was observed during the rainy season (n = 4706; 74.32%) than in the dry season (n = 1626; 25.67%), especially in peri-urban (93.83%) and urban areas (96.81%). This implies that when active monitoring of arbovirus vectors is enforced, seasonal surveillance must be considered as the densities of mosquitoes vary with varying seasonal factors.\u003c/p\u003e\n\u003ch2 id=\"_Toc187767820\"\u003e\u003cstrong\u003e4.3.4 Presence of arboviruses in the mosquitoes\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll sentinel sites for Karonga showed visible white bands during agarose gel electrophoresis, as all bands were along the positive control after reverse transcription PCR (RT-PCR) for both Chikungunya and Dengue. All sites were sensitivity to CHIKV E1 recombinant and not CHIKV FLY, which means that all the sites in Karonga had Chikungunya virus class E1, not the CHIKV fly, as both primers were used (Mavale et al., 2012; Cho et al., 2008). However, similar studies were carried out by Joannides et al., 2021, who wanted to find species composition and risk of arbovirus transmission in some parts of northern Ghana, and did not yield any positive results for arboviruses after RT.PCT from the 75 pools of \u003cem\u003eAedes\u003c/em\u003e mosquitoes. The findings of this study are, however, more consistent with those from the Kyera district, which is to the north of the Karonga district. In their findings from an entomological study carried out in Kyera town, Kajunjumele, Ipida, Matema, and Njisi villages from April to June 2015 (almost the same months as our study), Bisimwa et al., 2016; Bisimwa, N P, Angwenyi, S, Kinimi, E, et al., 2018 found arboviruses in 24 pools ofAedes mosquitoes from 480 Aedes mosquitoes collected. Arboviruses were detected in nine pools (37.5%), including alphaviruses (eight pools) and flaviviruses (one pool). None of the samples were positive for bunyaviruses. Chikungunya virus (CHIKV) was detected in six (75%) alphavirus-positive pools that were collected mostly in areas where rice cultivation was common. However, this study focused only on Dengue and Chikungunya, and all site samples were positive for flavivirus (CHIKV) and alphavirus (DENV). The findings of this study may indicate that people from these areas are equally exposed and at risk of arboviral transmission, similar to their counterparts in the neighboring district in Tanzania. This directly correlates with the human serum survey that isolated both chikungunya and dengue in patients who presented with febrile conditions at the facility in Karonga, Lilongwe, and Blantyre districts.\u0026nbsp;\u003c/p\u003e"},{"header":"CHAPTER FIVE CONCLUSION AND RECOMMENDATION","content":"\u003ch2\u003e\u003cstrong\u003e5.1 \u0026nbsp; \u0026nbsp; \u0026nbsp;Conclusions\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThis study confirms the presence of \u003cem\u003ethe Aedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e mosquito, which is a known vector for arboviruses that cause dengue fever and Chikungunya disease. Due to limitations in reagents, the study has only isolated dengue and chikungunya, but the presence of the competent vector shows that the population is also at risk of other arboviruses such as Zika. This study further complemented the human sero study that already established the presence of arboviruses in the human serum; thus, it has linked the transmission to humans due to infected vectors.\u0026nbsp;\u003c/p\u003e\n\u003ch2 id=\"_Toc187767824\"\u003e\u003cstrong\u003e5.2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Recommendations\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThere is a need for both habitats for breeding of these vectors must be taken care of if we are to control the spread of arboviruses. However, the other weather parameters were not critically analyzed and can be recommended for further studies to ascertain where rainfall and other weather parameters have a direct impact on the abundance of mosquitoes. Another area for further study could be the link between breeding sites and the abundance of different species.\u003c/p\u003e\n\u003cp\u003eI would recommend that further studies be carried out on the linkage between the vectors and human serology throughout the district of Karonga, and institute further serological studies as well as entomological studies that will further describe the disease burden in the district and immediately institute some preventive measures for the district. These findings would be shared with the Public Health Institute of Malawi (PHIM) and all concerned stakeholders to trigger the establishment of monitoring and surveillance programs on arboviruses before the district and country experience outbreaks of dengue and chikungunya, which have debilitating effects on the health and economy of the country. I would recommend more sentinel surveillance sites to be established throughout the country to establish the burden in the Karonga\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDC:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Centers for Disease Control and Prevention\u003c/p\u003e\n\u003cp\u003eCHIKV:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Chikungunya Virus\u003c/p\u003e\n\u003cp\u003eDENV:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dengue Virus\u003c/p\u003e\n\u003cp\u003eGIS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Geographical Information System\u003c/p\u003e\n\u003cp\u003eHMIS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Health Management Information System\u003c/p\u003e\n\u003cp\u003eIDSR:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Integrated Disease Surveillance and Response\u003c/p\u003e\n\u003cp\u003eITCZ:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Intertropical Convergence Zone\u003c/p\u003e\n\u003cp\u003eMEIRU:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Malawi Epidemiology and Intervention Research Unit\u003c/p\u003e\n\u003cp\u003eMLW:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Malawi-Liverpool Welcome Trust Clinical Research Programme\u003c/p\u003e\n\u003cp\u003eODK:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Open Data Kit\u003c/p\u003e\n\u003cp\u003ePCR:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQGIS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Quantum Geographical Information System\u003c/p\u003e\n\u003cp\u003eRNA:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ribonucleic Acid\u003c/p\u003e\n\u003cp\u003eSDGs:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Sustainable Development Goals\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWHO:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;World Health Organization\u003c/p\u003e\n\u003cp\u003eZIKV: \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Zika Virus\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eI, the undersigned, hereby declare that this thesis is my original work which has not been submitted to any other institution for similar purposes. Where other people\u0026rsquo;s work has been used acknowledgements have been made as reference in the document.\u003c/p\u003e\n\u003cp id=\"_Toc187767764\"\u003e\u003cstrong\u003eDEDICATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo my family members who endured my absence during my study and to all those who have dedicated their lives to medical entomology.\u003c/p\u003e\n\u003ch3 id=\"_Toc187767765\"\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eI sincerely thank my supervisors, Associate Professor Dylo Pemba and Dr. Themba Mzilahowa, for their effort, valuable time, guidance, and mentorship. In addition, I would like to thank Dr. Steve Gowero for providing the much-needed guidance during proposal development and laboratory work at Chancellor College. Special gratitude should go to Dr. Chigwechoka and Mr. Yohane Kazembe for dedicating their precious time to helping me during laboratory analysis.\u003c/p\u003e\n\u003cp\u003eI am also grateful for the support provided by Mia Crampin and Associate Professor Steffen Geiss, formerly of the Malawi Epidemiology and Intervention Unit (MEIRU) in Lilongwe and Karonga, who funded my accommodation and stay at Chilumba. I am sincerely grateful for the dedication and leadership of Jullita Malava and her team at MEIRU in Karonga. I am grateful to the research assistants and the data team who were there to offer support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe fieldwork was possible because of good coordination with the District Health Office team and timely transport provision by MEIRU team. 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Adult mosquitoes were trapped using oviposition traps (ovitraps), and aquatic stages were sampled using nets and/ or scoops and were subsequently reared to the adult stage. \u003cem\u003eAedes\u003c/em\u003e \u003cem\u003eaegypti\u003c/em\u003e was prevalent in all the six study sites. However, the study did not find \u003cem\u003eAedes albopictus,\u003c/em\u003ewhich is another known potential vector. The average \u003cem\u003eAedes Aegypti\u003c/em\u003e densities per site were: Hara 82 (15.7%), Iponga 144 (27.6%), Kaporo, 165 (31.6%), Karonga Town 128 (24.5%), and Kayelekera 3 (0.6%). There was an increase in density going further north of Karonga which borders Tanzania. Polymerase Chain Reaction (PCR) showed that all sites had dengue (DENV) and Chikungunya (CHIKV) viruses, implying that in sampling sites, communities are at direct risk of dengue, Chikungunya infection, and other arboviruses. Further studies are required to fully understand and characterize the extent of arboviruses among local communities and the role of \u003cem\u003eAedes aegypti\u003c/em\u003e in their transmission. There is an urgent need to set up the laboratory platforms, monitoring, surveillance and control systems for nation by the Public Health Institute of Malawi (PHIM) which is responsible for disease surveillance and response before arboviral diseases such as Dengue, Zika, Chikungunya, and others become epidemic and a major public health problem in the country.\u003c/p\u003e","manuscriptTitle":"Asssessing the potential of Arboviral Transmission in Karonga District, Northern Malawi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 05:30:18","doi":"10.21203/rs.3.rs-6883143/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d6dea33-541c-4133-bd1a-3ec8c5f290a4","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49975357,"name":"Virology"},{"id":49975358,"name":"Entomology"}],"tags":[],"updatedAt":"2025-06-16T05:30:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 05:30:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6883143","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6883143","identity":"rs-6883143","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}