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Targeting the mosquito

Research seeks alternatives to fight dengue carrier

cdcResistance: larvae remain immune to insecticide used years beforecdc

In late November, the Ministry of Health announced a potentially optimistic result in the fight against dengue fever: there are a lot fewer areas under imminent risk of new outbreaks of the disease. According to a survey carried out by the Ministry in the last week of October and the first week of November, 3.8 million Brazilians live in regions vulnerable to the spread of dengue, vs. 10.4 million in the same period for the previous year. Despite the lower number of critical areas, reports estimate that 32 million people live in regions still subject to some risk, especially in the northern and northeastern regions of Brazil. Gerson Penna, the Ministry’s National Secretary for Health Surveillance, believes that lower volume in critical areas is a direct result of campaigns to increase the population’s awareness and of state and municipal administration initiatives to eradicate the transmitter of the disease, the sleek, stripe-legged Aedes aegypti mosquito.

Up to now, however, the initiatives have been insufficient to avoid a 40% increase in the number of dengue cases over the last year. The disease causes a high fever plus muscle and joint pain, and can, in some cases, cause death. From just January to September of this year 481,316 people were infected with one of the three types of dengue found in Brazil, of which 1,071 resulted in dengue hemorrhagic fever. There were 121 deaths. In 2006, 345,922 cases of common dengue fever were reported, plus 682 of hemorrhagic fever; there were 76 deaths. “We must neither slacken our control nor discard the possibility of a new dengue epidemic”, stated Penna. “If we ease up now, the situation may deteriorate in just one week. We must be unwavering in the fight to eradicate breeding grounds of the transmitter mosquito”, concludes Penna.

Given how difficult it is to eliminate the transmission of the disease, which infects some 50 million people around the globe per year, researchers from a number of Brazilian institutes work in partnership with a team of the Ministry of Health’s National Dengue Control Program (PNCD), in pursuit of more effective control of the Aedes aegypti population. Entomologists, physicians, mathematicians and epidemiologists have combined their efforts to better understand the mosquito’s characteristics and behavior, identify alternative and more efficient chemical compounds to kill Aedes aegypti larvae or adult insects, and to find more effective means of monitoring the epidemics. They are also working on the development of vaccines capable of simultaneously protecting individuals against the four virus serotypes (see article on page 46). We must work very hard to contain the proliferation of dengue in Brazil and make sure that the Serotype 4 dengue virus, the most lethal, does not enter the country, as this strain is already found in some Latin American countries.

As there is no effective way of fighting the virus, the solution is to treat the disease’s symptoms and focus on eradicating the mosquito, whether in larva or adult form. Some of the results obtained to date show that even though the guidelines for dengue control programs are national, the initiatives to be adopted must be based on each region’s reality or, ultimately, on each municipality’s reality. “There are almost 5,600 realities in Brazil”, states biologist Denise Valle, from the Arthropod Carriers Physiology and Control Laboratory of the Oswaldo Cruz Institute (Fiocruz), in Rio de Janeiro, in reference to the 5,564 Brazilian municipalities.

The dengue trail
One of the reasons why dengue control should be regional is the limited mobility of the Aedes mosquito. Throughout its life cycle of about ten days, the mosquito does not fly much further than 500 meters from where it is born. Consequently, Aedes populations in remote areas, one hundred or even two hundred kilometers apart, may be quite distinct genetically speaking, with different susceptibility to insecticides and larvicides and also to the four dengue virus serotypes. Fiocruz entomologists Ricardo Lourenço de Oliveira and Magda da Costa Ribeiro studied the various genetic specimens of the Aedes aegypti in locations with varying occurrences of dengue and population densities in Brazil’s south and southeast.

Oliveira and Magda had hoped to find out how mosquitoes colonized others areas. “We wanted to discover the dynamics of dengue where dispersion of the mosquito is concerned”, stated Magda. People believed that Aedes could catch a ride in cars, buses, trains or planes and travel large distances. In order to find out whether mosquitoes in fact used the same transportation means as human beings, researchers used egg traps in eleven municipalities and major highway intersections that link the states of Minas Gerais, Espírito Santo, Rio de Janeiro, São Paulo and Rio Grande do Sul, in order to capture Aedes specimens in the dry and the rainy seasons. Researchers felt it was important to compare the southeast to the south because, when the research was being conducted, in 2002 and 2003, no dengue cases had been reported in the states of Rio Grande do Sul and Santa Catarina.

Magda analyzed the genetic characteristics of each mosquito population and noticed that they were differed. Detailed in an article published in the American Journal of Tropical Medicine and Hygiene, in the August 2008 edition, this result shows that mosquitoes practically do not hitch rides, at least not in these regions of the country and during the period analyzed. In specific locations, mosquitoes migrated less during the rainy season, when pools of water were common and females needed to fly only a short distance to find a good breeding ground for their eggs. During the dry season they had to fly further, but never quite far enough to justify the propagation of the disease from one state to another.

If the mosquitoes do not fly far, how does dengue spread? Probably through people who have been infected with the virus. Aedes is a fundamental mechanism in transmitting the dengue virus from one person to another, given that dengue fever does not spread by contact or air. But the virus is transported from one city to another or  to other states by contaminated people who travel during the transmittable phase of the infection, which lasts about one week. This form of dissemination of the virus is significant because of how easy it has become for people to travel long distances by car or plane in a short amount of time.

At present, Aedes aegypti might not hitch a ride to travel over large distances, but that was not the case in the past. People believe that the original mosquito came from Africa and arrived in Brazil in the seventeenth and eighteenth centuries aboard slave ships and spread throughout the American continent, with  the exception of Canada. In Brazil, it was exterminated in 1955 (and subsequently in other countries on the continent) as result of a Rockefeller Foundation campaign which began in the early twentieth century founded by  and maintained for several decades by the Pan-American Health Organization. Years later, however, the mosquito was back.

cdcThe USA in the twenties: workers draining swamp to control the reproduction of the dengue carriercdc

Entomologist José Eduardo Bracco, from the Bureau of Prevention of Epidemic Diseases (Sucen), which is part of the São Paulo State Health Bureau, and currently a guest researcher at the School of Public Health at the University of São Paulo, investigated the genetic similarities between Aedes aegypti populations from five countries in the Americas (Brazil, Peru, Venezuela, Guatemala and the US), three countries in Africa (Guinea, Senegal and Uganda) and three countries in Asia (Singapore, Cambodia and Tahiti). Based on the results, two theories were developed to explain the return of the disease in the Americas, especially in Brazil: some of the specimens of the mosquito survived the attempt to eradicate the species and returned to spread their larva throughout the country or they returned to Brazil from some neighboring country, such as Venezuela or the US, which were unable to eliminate the mosquito altogether.

In this study, which published in the August edition of Memórias do Instituto Oswaldo Cruz and carried out along with Ricardo Oliveira, from Fiocruz, and Maria Anice Sallum and Margareth Capurro, from USP, Bracco also concluded that an Asian line of the Aedes aegypti mosquito arrived in Brazil in the eighties, possible as result of the Brazil’s increased trade with Asia.

Hidden focal points
Since the reappearance of dengue in Brazil, the key strategy employed by Brazilian cities to control the mosquito’s proliferation has been to have homes inspected by health agents in order to identify potential breeding grounds and to eliminate Aedes aegypti points of reproduction. When breeding grounds are identified, the health agents apply insecticide to eliminate the larvae or the actual mosquitoes. This, however, is neither the only strategy, nor the most effective. In November an inspection agent, upon examining an upper middle-class neighborhood in the city of Campinas, inner São Paulo state, stated that he found a few cans with Aedes larvae. After completing an inspection where he did not find any breeding grounds, he was unable to explain why the house contained so many adult Aedes aegypti mosquitoes. Alvaro Eiras, a biologist from the Federal University of Minas Gerais has an answer: “In the dry season the mosquitoes reproduce in underground water galleries and in sewers.”

According to Eiras, just inspecting for dengue larvae is ineffective, as this is an outdated procedure, being a measure adopted in the twenties to combat yellow fever and that has never been updated. One of the reasons why this strategy is ineffective is because the amount of larvae does not always correspond to the concentration, at a given point in time, of adult insects in the reproductive phase, when females are roaming for the blood needed for the development of their offspring.  To have a better grasp of the problem in real time, Eiras developed a trap which he dubbed MosquiTRAP, which captures both larvae and adult mosquitoes (see Pesquisa Fapesp no. 131). A one-liter black plastic cylinder, it releases a chemical compound that attracts females who are looking for a place to lay their eggs. They enter the trap and end up stuck to an adhesive card. The method is efficient because adult mosquitoes are a better indicator of the infestation rate; it also enables researchers to identify the species of mosquito that live in a given area by simply examining the trap; the larvae can be identified in a laboratory using a process that takes about two weeks.

In a test conducted from March to June 2003 in the district of Itapoã, city of Belo Horizonte, Eiras compared the effectiveness of the MosquiTRAP with a trap that solely collected the insect’s eggs (egg trap) and with visual inspection of potential breeding grounds. The egg trap detected the presence of Aedes aegypti during the 17 weeks of the study, whereas the MosquiTRAP captured specimens of the mosquito in 13 of the weeks. Visual inspection, however, only identified larvae during two weeks of the study, according to results published in the beginning of the year in the Neotropical Entomology journal. Even though the egg trap turned out to be more sensitive in detecting Aedes aegypti eggs, MosquiTRAP is more precise as it captures adult females, which are a better indicator of infestation, as well as other mosquito species, such as the Aedes albopictus¸ which transmits other viruses.

Eiras transferred his technology to a company in the state of Minas Gerais, Ecovec, which has already successfully implemented the intelligent dengue monitoring system in 15 Brazilian municipalities, including large cities such as Vitória and Belo Horizonte. The inspection agent visits the houses where the traps are placed, in general 16 per square kilometer, and if an Aedes aegypti specimen is identified, the agent inputs the date and location of the insect on his/her mobile phone, data which is immediately transmitted to the company and posted on the Internet. The system generates maps of the municipality with color-coded areas – red where there is a higher mosquito density and green where no insects were found. Health officials have access to this data and can therefore focus their efforts on higher risk areas. “Something that for us is practically immediate takes one month for the Ministry of Health”, compared Eiras. “And in the meantime the mosquito continues to breed.”

The UFMG biologist stated that the infestation levels have been falling in the cities that have adopted this monitoring strategy. The results seem so promising that the Ministry of Health showed an interest in testing the MosquiTRAP to see whether it could be used nationwide. At the end of 2006, MosquiTRAP received the Tech Museum Award as one of the world’s top five inventions in the health sector.

cdc Aedes aegypti eggs: Feasible even after months with no contact with watercdc

Counterattack
According to Eiras, more effective monitoring of mosquito populations is essential to avoid indiscriminate use of insecticides, to which Aedes is becoming resistant. “We lose insecticides faster than we develop new ones”, stated Denise Valle, from Fiocruz, a member of the National Network for Monitoring Aedes aegypti Resistance to Insecticides (MoReNAa), the largest insecticide resistance monitoring program worldwide, which assists in the control of the dengue carrier in Brazil. From 2001 to 2004, its laboratory assessed mosquito populations from several different Brazilian municipalities in the states of Alagoas, Pará, Rio Grande do Norte, Sergipe, Goiás, Rio de Janeiro and Rio Grande do Sul. In many of the towns, the mosquito was no longer susceptible to the effects of temefos, an organophosphate larvicide widely used in the domestic market from 1967 to 2000. Although temefos has been replaced by piretroid insecticides to fight the adult insect and by bioinsecticides, made from Bacillus thuringiensis, to fight the larvae, resistance to temefos persisted until up to three years ago. While examining the biochemical mechanisms that allow Aedes to gain resistance to insecticides, Denise identified in all regions analyzed an increase in the larvae of the glutathione-S-transferase (GST) activity, which neutralizes the effect of not only of temefos but also of piretroids. “This increase coincides with the period when the use of piretroid began in Brazil; however we are still unable to confirm whether the enzyme is actually responsible”, explained Denise. These results, published in the September issue of the American Journal of Tropical Medicine and Hygiene, are alarming because they indicate that the use of a number of insecticides is still compromised.

In their pursuit of ways to fight the mosquito, some research groups have centered their efforts on developing insecticides with plant-based active ingredients. “We must exploit Brazil’s biodiversity”, stated Antônio Euzébio Goulart Sant’Ana, from the School of Biotechnology and Chemistry at the Federal University of Alagoas. His team tested the larvicide efficiency of extracts from 51 species of Brazilian flora against the Aedes aegypti; the team also tested the toxic effect of these extracts on other animals, to ensure that the selected compounds did not harm the environment. According to an article published this year in the journal Bioresource Technology, the most promising results were obtained from the leaf extracts of the araticum bush (Annona glabra and Annona crassiflora). “Now we are refining the most effective compounds”, stated Sant’Ana. According to the researchers, there is yet another advantage in developing vegetable-based insecticides: this could result in an additional market for farmers who grow these plants.

Onilda Santos da Silva, a biologist from the University of Southern Santa Catarina, is developing other alternatives. She analyzed the effect of seeds and oil of andiroba (Carapa guianensis) on Aedes. These seeds and oil are used by local indigenous communities in the Amazon region and they seem capable of eliminating insect larvae resistant to temefos, as detailed in articles in the Journal of the American Control Association. Onilda has also achieved good results with the chinaberry (Melia azedarach) and the Pinus caribaea pine tree and stated that the development of the larvicide should be concluded by the end of next year. Even if an effective vegetable-based larvicide is obtained, it will be necessary to produce it in large scale first before it can be used to fight dengue. The Ministry of Health, which has been investing in new insecticides, also recommends that they be approved for use in drinking water.

From theory to action
However, more effective insecticides are not enough to contain the mosquito that has been frustrating health officials throughout Brazil. It has become increasingly evident that we need to take action not only against larvae but also against adult insects, and that we must eliminate potential breeding grounds, in keeping with the work carried out in Singapore, in Southeast Asia, by a team led by researcher Eduardo Massad, from the USP School of Medicine. By using a mathematical model that takes into account several different parameters (number of people infected, recovered or immune to dengue; number of mosquitoes susceptible to the virus; and infected insects or eggs), Massad built a realistic picture of how the disease spread throughout Singapore, a country with roughly four million inhabitants in southern Malaysia.

Based on statistics from the 2004 and 2005 epidemics, Massad simulated the best strategies to fight dengue in Singapore. The results, presented this year in the Epidemiology and Infection journal, reveal that two simultaneous activities were required: to cut the population of mosquitoes and larvae in half, while also eliminating the number of breeding grounds. And an ongoing effort to control the transmission of the disease is not required. Control activities may be carried out once every five weeks. The model also confirms that once the epidemic has materialized, the best course of action is killing adult mosquitoes, though fighting larvae and eliminating breeding grounds is essential to avoid the resurgence of the dengue mosquito.

Even though the Singapore government embraced the described measures, a new epidemic of dengue broke out two years ago, at a time when the disease seemed to be under control. “Transmission, which previously occurred inside homes, started to take place outside people’s houses”, explained Massad. So authorities needed to change their strategy, given that house-to-house inspections, as is carried out in Brazil, were no longer effective. The government now began to monitor the mosquito-infested regions, checking which areas had reported cases of dengue and fighting the epidemic through a combination of larvicides applied to breeding grounds, insecticides used against adult mosquitoes and a quarantine to reduce contact between the ill and the healthy population. These measures worked. They were implemented at a peak of 697 dengue cases in the last week of September, and in about two months they cut the incidence of the disease to around one hundred new cases per week.

However, there is still a lot of work to be done. This year, although the control measures were maintained, there are far more dengue cases than the model forecasted. “We are researching which factors may be responsible for this increase”, stated Massad, who has a hypothesis: last year, a serious fire in Indonesia killed a large portion of the mosquitoes. It is possible that the survivors became stronger. Given these results, the Ministry of Health showed some interest in using the Singapore experience in Brazil, said Marcelo Burattini, a member of Massad’s team and coauthor of the Singapore study. Burattini attended a meeting with the national and the São Paulo coordinators of the struggle against dengue in November.  Statistics are lacking  here in Brazil detailing where the mosquitoes are located and the areas where the disease is concentrated, in addition to other parameters that are needed in order to simulate the best strategies to be adopted. “One of next year’s priorities is to improve the entomologic surveillance parameters through new capabilities such as traps for the capture of adult mosquitoes”, said Burattini.

Even so, it will not be easy. “There is a difference between what is simulated in a research environment and the actions that are put into place in reality”, commented the researcher from USP. Additionally, the actions recommended by the PNCD coordination must be adapted to the different Brazilian regions. And what is even more complicated: if they are not carried out with the same efficacy in all municipalities, the strategies lose their effectiveness. As Denise Valle puts it, it is an arduous job that has to be carried out one step at a time.

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