Cristina Valdovinos

Nkemjika Ugonabo

23 May 2008


Assessing the impact of treatment of septic tanks with expanded polystyrene beads on Aedes aegypti larval and adult mosquito emergence



Brazil is currently experiencing one of the worst outbreaks of Dengue and Dengue Hemorrhagic Fever (DHF) in recent history. This year alone, the Brazilian state of Amazonas has experienced nearly 10 times the number of cases compared to the same period last year, suggesting a more severe evolution of the illness (WHO, 2008). Alarmingly, the Brazilian Ministry of Health recognizes that the country's hospitals do not have the capacity to respond to these outbreaks (WHO, 2008). Brazil's current approach of mass spraying following outbreaks has not proven successful. To date control strategies have been reactive, focusing only on controlling the adult mosquito (WHO, 2002).  This has necessitated the search for alternative control measures to contain the Aedes egypti mosquito vector at other stages of the arthropod’s lifecycle. This study seeks to investigate how both larval and adult mosquito control strategies can be implemented as means of reducing the adult mosquito population.

Specific Aims

 This study, which will be conducted in Rio de Janeiro, Brazil will evaluate the efficacy of treatment of household septic tanks with Expanded Polystyrene Beads (EPSB) in reducing Aedes aegypti emergence.  The intervention households will receive one-time treatment of septic tanks with EPSB beads and the control group will receive no treatment. We will determine the percentage difference of adult and larval populations in intervention and control groups at various intervals by conducting larval and adult mosquito surveys of household septic tanks. The difference in mosquito population rates in the intervention and the control groups will be compared. We predict that treated households will demonstrate a significant reduction in larval and adult mosquito populations and that control households will not experience any significant reductions.


Dengue is considered to be one of the most important vector-borne diseases in the world and the emergence of this disease in the past three decades has become a major public health concern.  There are over 50 million dengue infections each year and 2.5 billion people live in areas where dengue viruses can be transmitted. The disease is endemic in more than 100 countries (WHO, 2007).

The emergence of dengue has been most dramatic in the Americas and Brazil has been the hardest hit (CDC, 1995). In 1973, Brazil declared that the Aedes aegypti mosquito, the principal vector of dengue, had been eradicated. Victory was declared prematurely however. Ae. Aegypti reappeared within three years and by 1986, Brazil experienced the first epidemic (WHO, 1995). From that point on the dengue epidemic has continued unabated. Brazil accounted for nearly 70% of the more than 3 million reported cases of dengue fever in the American regions this decade (Siqueira et al., 2005).

This year controlling the dengue epidemic is as urgent as ever. In April 2008, the WHO reported a significant increase in the number of cases of dengue in several Brazilian states compared to the same period in 2007: Amazonas (9.8 times), Rondônia (5.3 times), Sergipe (4.7 times), Bahia (3.4 times), Rio Grande do Norte (2.8 times), Pará (2.5 times), and Rio de Janeiro (2.2 times) (WHO, 2008). The unexpected rise in the number of cases has taken a toll on the Brazilian public health infrastructure, particularly in the southern municipality of Rio de Janeiro where there have been more than 76,000 cases this year alone. The Brazilian Ministry of Health mobilized 600 health professionals to the federal hospitals of Rio de Janeiro to support dengue activities (WHO, 2008).

Although the reasons for the emergence of dengue are not entirely understood, there is agreement that increased air travel, global population growth, substandard housing, crowding and deterioration water and waste management systems resulting from unplanned urbanization have created ideal conditions for the dispersal of the Ae. egypti vector and the dissemination of the virus (Gubler, 1998). It has also been suggested that climate change can modify the mosquito’s geographic distribution and increase the transmission of dengue (Hopp and Foley, 2001).

The Dengue viruses

Dengue is a viral infection transmitted from person to person via the bite of an infected female mosquito. There are four related, but antigenically distinct dengue viruses: DEN 1-4. These viruses are part of the Flavivirus genus, Flaviviridae family. The viruses are small, spherical, and about 11,000 base pairs long (Gubler, 1998). Infection with one of strain of the virus does not confer immunity to the other three. People living in hyperendemic areas can therefore be infected by more than one virus type (Gubler, 1998).

The Vector: Aedes Aegypti

The main vector of dengue is the female Aedes aegypti mosquito, which is also the principal vector of yellow fever and Japanese encephalitis. Ae. aegypti mosquitoes preferentially feed on humans, and require a blood meal for oviposition. The mosquitoes prefer to feed indoors during daylight hours. They are holometabolous, undergoing complete metamorphosis through egg, larval (with four substages), pupal and adult stages (Hopp and Foley, 2001). Climatic variables such as temperature and precipitation influence the mosquito’s development and survivorship (Gubler, 1998).

Single elongated or ovoid eggs are laid either at or above the waterline, or on the surface. The mosquitoes prefer to lay eggs in domestic and peri-domestic artificial containers including tires, water drums, and septic tanks (Gubler, 1998). Hatching of eggs occurs after several days to weeks, is temperature-dependent and occurs quickly when the water level rises (Wild Life Info, 1998). All four larval stages are aquatic. The mosquitoes larvae molt four times collectively require a minimum of four days to complete their development but take an average 6-10 days. The larvae usually mature within two weeks (Wild Life Info, 1998).

After the fourth molting, the pupa emerges from a T-shaped hole in the back of the last larval skin. Pupae are non-feeding, aquatic and motile. They have paddle-like, oval extensions attached terminally to the abdomen to move up and down in the water. This stage is usually short: from a few hours to two days. The pupae release the adult through a split in the back of the pupal cuticle and the pupal case floats on the surface of the water. The wings are fully expanded and hardened after about 24 hours (Wild Life Info, 1998).

The mosquitoes live on average one or two months. Female adult mosquitoes require a blood meal to oviposit. This cycle requires two or more days for the female to digest the blood, lay a batch of eggs, and seek another blood meal. This can be repeated many times in a female's lifetime. When the female mosquitoes feed, the proboscis form a tube and the stylets penetrate the skin of the animal and form a small duct through which saliva is injected into the wound; they also act as a canal through which liquid food is ingested. Only one mating is required to fertilize her lifetime egg production (Wild Life Info, 1998).

Figure 1: The Aedes Aegypti Life Cycle (Hopp and Foley, 2001)


There is an enzootic dengue transmission cycle in the forest involving Aedes mosquitoes and lower primates in Africa and Asia but because there is rarely movement of the enzootic cycle into urban areas, the most important cycle is the urban transmission cycle. Because of high viremia resulting from dengue infection of humans, the viruses are efficiently transmitted between mosquitoes and humans without the need for an enzootic amplification host (Whitehead et al, 2007).



Figure 2: The Sylvatic and Urban Dengue Transmissions Cycles (Whitehead et al., 2007)

Below the urban transmission cycle is divided into four phases:

A : Person infected with dengue has high viremia for five days along with a host of other symptoms.

B: An uninfected female Aedes aegypti mosquito feeds on infected human host and acquires dengue virus. The virus replicates within the mosquito midgut and salivary glands during an extrinsic incubation period of eight to twelve days.

C: The mosquito then feeds again on a susceptible host and transmits the virus from its salivary glands to him or her, as well as to every other susceptible person the mosquito bites for the rest of its lifetime (CDC, 2002).

D: In the second person, the virus undergoes an intrinsic incubation period of 3 to 14 days (on average 4 to 7 days) accompanied by the onset of symptoms, which may last between three to 10 days. During this period, the virus circulates in the peripheral blood (CDC, 2002).

Symptoms of dengue include headache, back pain, joint pain, myalgias, anorexia, and a variety of nonspecific signs and symptoms, including nausea, vomiting, constipation and rash. The fever lasts between 2 to 7 days. There is no treatment for dengue as the infection is usually self-limiting and is rarely fatal (Gubler 1998). However, a fraction of cases progress to Dengue DHF. DHF is characterized by increased vascular permeability and hemorrhagic manifestations including skin hemorrhages (Gubler, 1998). DHF can progress to Dengue Shock Syndrome, which is characterized by leakage and loss of blood volume. It has been suggested that virus strain can determine if an infection will progress to DHF. Pre-existing anti-dengue antibodies, host genetics, and young age may also play a role. There is also higher risk in hyperendemic areas. (Jacobs, 2005).

Current Approaches to Combat Dengue in Brazil

Because there are no treatment or vaccines available for the dengue viruses, there is agreement that elimination or significant reduction of Ae. aegypti populations is an effective and proven method for disease prevention (Morrison et al. , 2008). Vector control is the only means of interrupting the transmission cycle. To date, focus in Brazil has been to control at the vector at the adult stage, that is, to mitigate rather than to prevent the outbreaks. Brazil’s plan of dengue control activities (PIACD) includes vector control by mass spraying, motivating families to take responsibility for the maintenance of domestic environment free from possible vector breeding sites and strengthening epidemiological and entomological surveillance to expand the capacity for outbreak foresight and early detection (WHO, 2002). Unfortunately, as the current outbreak illustrates, the current approach is largely ineffective due in part because it waits for the mosquitoes to emerge before eliminating them. As de Andrade Medronho at the Federal University in Rio de Janeiro expressed: “[Brazil] must be humble enough to admit that the current strategy has failed (at least in the large Brazilian cities) and have the courage to change it” (de Andrade Medronho, 2008).

We propose that prevention of oviposition coupled with larval and vector control is a more effective strategy to combat dengue than the current focusing solely on the adult mosquito. Larva cannot transmit disease and are less mobile than adult mosquitoes making them a better target for reduction and elimination. Moreover, preventing oviposition and intervening at the larval stages prevents the emergence of mosquitoes that can later transmit the infection.

Expanded Polystyrene Beads: An Alternative Control Strategy

Previous trial studies have demonstrated the effectiveness of treatment of mosquito breeding sites with EPS beads in reducing mosquito emergence. In Kenya, Zimbabwe and Tanzania successful EPSBs trials have carried out against C. quinquefasciatus breeding in pit latrines, soakage pits, and septic tanks (Sivagnaname et al., 2004). In India, EPSBs have also been used on small scale for the control of A. stephensi and A. culicifacies in unused wells. In Malaysia a field trial of the use of EPSB to control the breeding of mosquito larvae in household septic tanks showed a 68% reduction in the breeding of Aedes albopictus (Chang et al., 1995). Much of the past scientific research on EPSB has focused on its effect on the control of A. albopictus and A. stephensi, which vector other diseases. There is little data on the use of EPSB to combat the breeding Ae. aegypti, but given the successful results with related species such a study is warranted. 

Our proposed study will explore the effectiveness of treatment of septic tanks (which contribute significantly to the maintenance of dengue virus endemicity) with EPS beads in reducing larval and mosquito populations.

EPSB Beads

Expanded polystyrene beads  (EPSB) are hard translucent, glass-like beads that can act as an effective, feasible and environmentally safe method of mosquito breeding control.  EPS beads are used to cover the water surface of mosquito breeding sites e.g. septic tanks.  The beads form a thick blanket layer that makes it extremely difficult for larvae to respire, prevent the emergence of adult mosquito, and create a dry surface unsuitable for oviposition (Sivagnaname et al., 2004).

The mechanism behind this is simple (Becker et al., 2003).  Unexpanded polystyrene beads contain an expanding agent that when heated rapidly expands the beads up to 30 times in volume (Becker et al., 2003). Beads can be easily heated with steam or boiling water. The simplicity is important for community education and implementation, as it provides a cheap and practical method that can easily demonstrated vector control method (Chang et al., 1995). EPS beads do not need frequent application since they remain on the surface for lengthy duration of time (up to four years) (Curtis et al., 1986). EPS beads represent an excellent advantage to other mosquito control methods like insecticides in that they have less of an environmental impact (Sivagnaname et al., 2004).

Experimental Design

Study area

The study will be undertaken in Rio de Janeiro, Brazil. Candidate households will be recruited with help from the Rio de Janeiro branch of the Center for Strategic Information in Health Surveillance. Households on the candidate list will be screened with pre-designed questionnaire to determine infestation rates and financial information. 200 households of similar socio-economic status and Ae. aegypti infestation levels will be chosen to participate (Chang et al., 1996).  Families will be compensated appropriately for their participation, as according to the Stanford Human Subjects Protocol.

Households selected will be randomly and equally divided into two groups:

Intervention Group (A):

EPBS, 2 millimeters in diameter, will be applied to both chambers of these septic tanks to form a 2-3 centimenters thick layer on the water surface (Sivagnaname et al., 2004). Septic tanks of houses in this group will be treated with the beads for 8 weeks.

Control Group (B):

Septic tanks of the houses in this group will not be treated.

Larval Survey

Using a long-handled dipper, septic tanks in both groups will be surveyed for mosquito larvae one week prior to EPSB treatment (Chang et al., 1995). Surveys will be repeated one week post-treatment and continued at biweekly intervals for 8 weeks.

Mosquito survey

Wooden framed, pyramidal nylon exit traps will be placed over the septic tanks to catch and sample emerging adult mosquitoes (Chang et al., 1995). Traps will be set over five randomly selected septic tanks in the treatment and control areas.  Each trapping round will last for 4 consecutive nights and trapping will be carried out one week before and one week after treatment, and then at biweekly intervals for 8 weeks.  Ae. aegypti in traps will be counted  once per week.


Results will be evaluated in two ways. In our intervention group, the impact of the treatment will be assessed by comparing difference in larval populations and adult emergence rates pre-and post-intervention (Chang et al., 1995). Then we will compare the reduction rates of the intervention group with the reduction in the untreated control group. This will confirm that the EPSB treatment is associated with the reduction. Percent difference in larval and adult populations will be measured and statistically analyzed for significance.

Implications for Vector Control

Although the proposed study does not directly measure the change in incidence of dengue fever, it is still provides very useful information because infestation of Ae. aegypti is directly correlated with dengue onset. It is logical that a decrease it one would lead to a decrease in the other.

By demonstrating the effectiveness of EPSB in preventing oviposition and reducing both larval and adult the Ae. Aegypti populations in septic tanks, we hope to provide a sustainable, cost-effective means to reduce the emergence of Ae. Aegypti from common breeding sites. The expected results would have major implications for Ae. Aegypti control in Brazil.  By evaluating  the effectiveness of this intervention, we can advance knowledge that  may help prevent future dengue outbreaks in Brazil.








Works Cited

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