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New attacks on dengue

Monitoring with a trap that attracts mosquitoes is one of the innovations, together with transgenic insects, for controlling the disease

LUCIANO ANDRADE MOREIRAGenes inserted into the genome of the Aedes work like antibodies against dengue, with a direct interference with the virusLUCIANO ANDRADE MOREIRA

The abundant summer rains and the high temperature form a dangerous combination that contributes towards the explosion in the population of the Aedes aegypti mosquito and the subsequent transmission of the virus of dengue, a disease characterized in its classical form by a high temperature, a headaches, and many pains in the body, but which rarely kills. More serious is hemorrhagic dengue, which, besides the classic symptoms, also causes bleeding, insufficient circulation and a fall in blood pressure, and may lead the victim to die. The disease affects more than a hundred countries in various continents and in the form of epidemics that repeat themselves. Estimates of the World Health Organization (WHO) indicate that between 50 and 100 million persons are infected every year, with a balance of 550 thousand internments and 20 thousand deaths as a result of the disease.

In Brazil, the picture is not very heartening. After facing an epidemic of dengue in 2002, with almost 800 thousand cases notified, specialist are afraid that a new outbreak may occur this year. “There is a risk of introducing serotype 4 (serotypes 1, 2 and 3 of the virus already exist in Brazil), considered the most lethal, which is to be found circulating in several countries of the Americas”, says Maria da Glória Teixeira, a professor from the Collective Health Institute of the Federal University of Bahia (UFBA) and the author of an epidemiological study about dengue. Up until now, no efficient remedy against the virus of the disease has been discovered. It calls for medical accompaniment and is treated with medicines that alleviate the symptoms, besides rest. Vaccines to fight dengue are being studied, but there is still a long road ahead, because to be efficient they will have to immunize people simultaneously against the four serotypes of the virus, which is from the flavivirus family.

With so many difficulties in overcoming the virus, the thing is to fight the mosquito, which also transmits yellow fever. In 2006, new ways of controlling the Aedes were presented, capable of preventing the proliferation of the mosquito. In the front line of the new technologies is a system for monitoring the mosquito that transmits dengue that uses traps, software and handheld computers (palmtops) to catch the insects and analyze the areas of risk. Inside the trap called Mosquitrap, a sort of black vase with water at the bottom that imitates the mosquito’s breeding ground, Atraedes is put, which is a synthetic substance that releases an odor to attract and to make pregnant Aedes females lay their eggs in the vessel. “The aromatic product was isolated from an infusion prepared with Panicum maximum grass”, says Professor Álvaro Eduardo Eiras, from the Biological Sciences Institute of the Federal University of Minas Gerais (UFMG), responsible for the development of the system called Intelligent Monitoring of Dengue.

On being attracted by the Atraedes, the insects go into the trap and get stuck to an adhesive card placed on the wall of the vessel. After one week, a count is taken of how many mosquitoes have been caught, a job carried out by health agents trained to recognize Aedes in its adult form. It is not necessary to take the insect to laboratory to be identified, as currently occurs with the larvae taken from vases and tires full of water. “By attacking the mosquito directly, we prevent the female from depositing its eggs. We are working on the control of the insect with traps because the transmission of the virus of dengue is done by the infected female”, Eiras says. When the female, which needs blood to mature its eggs and make the reproductive cycle continue, bites an infected person, the virus is installed and multiplies in the salivary glands and intestines. From then on, the insect remains infected for the rest of its 30 to 40 days of life, on average.

Mosquitrap carries out a control of the infestation by means of a handheld computer. The data on the quantity of mosquitoes caught in the trap is sent to a center that, in three hours, generates a precise map of the areas at risk of infestation with the disease. This map is available online for the health managers to know where to concentrate the actions for fighting dengue.

Based on the knowledge of the behavior of the vector, the team from Ecovec, a company created to develop and to market the product that arose in the university in Minas, which has Eiras as one of its partners, is preparing a project with local diagnoses, determining the number and the positioning of the traps necessary for guaranteeing the effectiveness of the monitoring. The data is updated on a weekly basis, which means 52 maps a year. “The cost is about 90% lower than the cost of the monitoring system for the larvae of the Aedes used in several countries, Brazil included, between four and six times a year”, Eiras says. The analysis of the larvae collected by the health agents on their home visits is done in the laboratory, which demands time and extra costs. Without mentioning that the use of insecticides to fight the Aedes does not take into account whether the mosquito is present or not.

Award-winning system
Ecovec’s method manages to detect the presence of the mosquito even at times of drought. So much so, that there is the possibility of the system beginning to be officially adopted in 2007 by the Ministry of Health as part of the National Program to Control Dengue, which received R$540 million from the federal government in 2006 for actions to fight and to control the vector. Last November, the system was chosen from amongst 280 initiatives from 58 countries to receive the Tech Museum Award, in the Health category, handed over in San José, in California.

The award is given for innovative technologies that benefit humanity and has the support of companies like Intel, Accenture, Microsoft, Agilent, Applied and HP. Bill Gates, of Microsoft, was paid homage at the same event. “In the speech that he made in the course of the award-giving, Gates said that the technology developed in Brazil to fight the vector of dengue is simple and genial to solve a complex problem”, Eiras reports.

LUCIANO ANDRADE MOREIRAThe city of Congonhas, in Minas Gerais, was the first to adopt the innovative system, over a year ago. “Since then, no more cases of dengue have been recorded in the municipality”, Eiras says. Frutal, also in Minas, and Vitória, the capital of Espírito Santo, are following the same path. The trap has now been sold to some countries, like Germany, Singapore, Panama, France and Italy. At the end of November, Ecovec received a visit from a representative of the government of the state of Queensland, in Australia, interested in adopting the system for the intelligent monitoring of dengue.

The price for implanting the technology depends on the size of the city and the degree of infestation by the mosquito. In the city of Congonhas, for example, with 45 thousand inhabitants, the system costs R$5,800.00 a month. The service provision contract includes about 200 traps installed, besides the aromatic substances, adhesive cards, palmtops and the whole process of processing the data and generating the maps of infestation, graphs and tables, with free access by the health manager to the Internet. Eiras stresses that the calculation has to take into account the savings made by fighting the mosquito before the mosquito installs itself. And he cites the case of the city of Uberaba, in the the southwest of Minas Gerais, which to control an epidemic of dengue spent R$1.3 million just to fight the mosquito, not counting the internments. “Had the city used our technology, the cost would have been much lower”, is the researcher’s comparison. A survey by the Public Health System, of the Ministry of Health, indicates that the treatment for dengue costs R$250.00 per person. In the case of internment, the expense is as much as R$3,500.00.

With the summer rains – which began to fall hard last November -, the specialist fear a new outbreak in 2007, similar to the one that occurred in 2002. “We have to advance in the question of controlling the population of vectors and in the technology we have available today, because the treatment of the focuses using larvicides is not being effective”, says Maria da Glória Teixeira, from UFBA, the main author of a study about the epidemiological, situation of dengue in Brazil and in the world, published in the Cadernos de Saúde Pública [Public Health Notebooks], of the Oswaldo Cruz Foundation (Fiocruz), of Rio de Janeiro.

“Brazil’s big urban centers have been the most affected, because of the high population density and deficient sanitation conditions”, the researcher says. According to the study, dengue is one of the world’s biggest public health policies, due to its great geographical expansion, complex clinical and epidemiological conditions, and, in particular, for the difficulties encountered in controlling it. In Brazil, three of the serotypes of the virus are circulating, types 1, 2 and 3, the last of which was introduced at the end of the 1990’s and in a mere three months it had already been disseminated to eight states, which shows the facility for the new strains to circulate with the multitudes that are moving about every day. Infection by one of them provides permanent protection for the same serotype and temporary partial immunity against the other three.

“Several studies done at UFBA show that the transmission and circulation of the virus is far more rapid than the current capability for fighting the vector”, the researcher says. While the combat against the larva is being waged, the females of the adult mosquito can be in the environment transmitting the virus. For this reason, Maria da Glória believes that the technology developed at UFMG may have a great impact on controlling dengue.

The researcher is working on the hypothesis that by reducing the population of the female adults, caught in the traps, there is a decrease in the transmission of the virus. To verify whether the assumption is correct, she presented a project to the Ministry of Health, in partnership with Eiras, which will be carried out in a large urban center, such as Salvador or Rio de Janeiro. The project provides for a comparison of the incidence of infections by the virus of the dengue between areas with traps installed and other areas of the same city where only the traditional treatment is done. “The study involves conducting seroepidemiological surveys in the population resident in the selected areas, which will make it possible to evaluate whether a reduction in the transmission of the virus of dengue will occur”, says the researcher.

Another front in the fight against dengue associates a traditional trap known as ovitrap, a biological larvicide and the use of GPS (a satellite-based geographical locator) to monitor the population of the Aedes. The project, which has the objective of developing an intensive model for controlling the mosquito without the use of chemical insecticides, has been carried out for two years in Recife and in its Metropolitan Region by a team coordinated by Lêda Regis, from the Aggeu Magalhães Research Center, a Fiocruz unit in Pernambuco, in partnership with the Secretariat for Health. The ovitraps are made of plastic and carry inside them an infusion of grasses. The traditional ones are very small, with a capacity for holding 300 to 500 milliliters of liquid. They have to be emptied every seven days, for the risk of the infusion turning itself into a breeding ground for mosquitoes.  “We adapted the ovitraps for them to be able to remain longer in the field and to reduce the operating costs”, says Lêda.

Biological larvicide
The adapted traps hold two and a half liters of the infusion of water and grass, and they all have inside three supports, where the eggs are deposited, instead of just one. Furthermore, to prevent the larvae from developing, a biological larvicide was added, made by Bthek Biotecnologia, a company from Brasilia, the main component of which is the Bacillus thuringiensis israelensis (see Pesquisa Fapesp No. 85). This bacterium produces a toxin that, when ingested by the larva, causes damages to the insect’s intestines, leading to its death. The addition of the Bti, the commercial name of the bioinsecticide, kills the larvae and, at the same time, works as a stimulant for the females to lay their eggs in the traps. The larvicide also makes it possible for the ovitrap to stay for over a month in the field.

LUCIANO ANDRADE MOREIRAOnce a month, health agents remove the supports to which the eggs are stuck, take them for counting in the laboratory, and renew the infusion of grass and larvicide. As the ovitraps have geographical references, all the information collected is put onto a map of the city. “It is a sensitive and effective instrument for detecting and monitoring areas with a high intensity of the Aedes mosquito”, Lêda says.

The ovitraps detected the presence of eggs, and consequently of female adults, in 88% to 96% of the properties in seven districts of Recife.  To illustrate the effectiveness of the trap, Lêda mentions that in a single ovitrap 8,900 eggs were gathered in the period of one month. In the same area, the level of house infestation, the method traditionally used, which is based on a visual survey of the larvae, was around 1%. “One of the experiments carried out resulted in gathering and burning over 10 million eggs and a 60% reduction of the population density”, Lêda says. The project is part of the Unified Support System for Detection and Accompaniment in Epidemiological Surveillance (Saudavel in the Portuguese acronym), which has as its partners, besides Fiocruz, the National Institute for Space Research (INPE), the Federal University of Paraná and UFMG.

The quest for alternatives to the chemical control currently used opens up other research fronts, such as the creation in the laboratory of genetically modified mosquitoes. One of the research groups, coordinated by Professor Margareth de Lara Capurro Guimarães, from the Biomedical Sciences Institute of the University of São Paulo (USP), constructed genes that work like antibodies against dengue, with a direct interference with the virus of the disease. “They recognize the viral particle and try to prevent the virus from reaching the mosquito’s salivary gland”, says Margareth.

The first stage for the creation of transgenic mosquitoes is the assembly of DNA sequences, inserted into the insect’s genome, to make it a less efficient vector. The genes of interest are then injected into the embryos, which are the eggs laid by the females to obtain genetically modified lineages. It is only after the birth of the larvae that it can be known whether the mosquitoes are actually transgenic. “We injected 2,500 embryos to get 20 families. The proof of being transgenic lies with the marker gene, inserted with the gene of interest chosen to fight the mosquito of dengue. The marker gene determines the production of a fluorescent protein and is only seen with a microscope. If the larva shows green or red eyes and bright spots of the same color on its back, it is transgenic.

The group from the Biomedical Sciences Institute is also researching into sterilization of the male by irradiation with cobalt. This biological control is already used in agriculture to fight fruit flies. “By making the males sterile in nature, there is a decrease in the population”, Margareth says.

The female of the Aedes has a sexual behavior that is similar to that of the fruit fly. It is faithful to a single companion. Accordingly, if it breeds with a sterile male, it will be incapable of procreating. The females of the Aedes lay, at five day intervals, the eggs resulting from a single fecundation. If she is infected with dengue, part of its offspring will also be infected. For this reason, when cases of dengue begin to be reported in a region, very rapidly they increase exponentially.

Lethal gene
Besides the direct interference on the virus, another line of research carried out at USP has as its basis an action mechanism that consists of putting a lethal gene into the mosquitoes that is expressed only in the females. “In the laboratory, we have a control for the expression of this gene to be kept switched off, and so we can get transgenic families”, says researcher Mauro Toledo Marrelli, from the School of Public Health. This control is done by putting a chemical repressor, like the tetracycline antibiotic, in the midst of the larvae.

In nature, only the males with the lethal gene would be released to mate with the wild females, which are going to produce lineages that do not get past the pupa phase, an intermediary stage between the larva and the mosquito. “This is a type of control to diminish the population that works as if it were a more specific insecticide, which does not pollute the environment”, Marrelli says.

ecovecSystem developed at UFMG uses a trap that imitates the breeding ground of the Aedes, with water at the bottom and a synthetic substance that attracts the pregnant females of the insect. On entering the trap, the mosquitoes are stuck to an adhesive and dieecovec

The studies are being done with Culex quinquefasciatus, better known by its popular name of skeeter or southern house mosquito. The same genetic construction can be used with Aedes aegypti and other mosquitoes, such as the one that transmits malaria. “The main target for the genetically modified mosquitoes is African malaria, which kills 2 million children a year”, Margareth says.

In Brazil, a research group coordinated by Luciano Andrade Moreira, from the René Rachou Research Center, of Belo Horizonte, which belongs to Fiocruz, obtained four transgenic lineages of mosquitoes of the genus of Aedes fluviatilis, a vector of avian malaria, transmitted by Plasmodium gallinaceum. Obtaining lineages means that, when they are at the adult stage, the modified mosquitoes will mate with others of the colony and will have offspring that will already be born with the genetic alteration. The gene responsible for blocking the malaria parasite is a protein called phospholipase A2, present in the venom of bees, capable of blocking the plasmodium, which is a protozoon (a unicellular organism) responsible for infection by malaria, when present in the mosquito’s intestines.

The study, which began two years ago and has the support of the World Health Organization (WHO), was started with the malaria in poultry, for being easier to obtain the complete cycle of this parasite and for the similarity of P. gallinaceum to Plasmodium falciparum, which infects humans. “The next step is to try to genetically transform the Anopheles aquasalis mosquito, a vector of human malaria in Brazil”, Moreira says. Although the breeding of genetically modified insects seems to be a promising alternative for facing up to the mosquitoes’ resistance to chemical insecticides, there is no way of foreseeing how long it will take for them to be released in nature, not least because more in-depth studies are needed to be sure that they will not cause an environmental imbalance. After that, it will still be necessary to obtain authorization from the National Technical Biosafety Commission (CTNBio), the entity responsible for approving genetically modified organisms.

Biotechnological traps, to start with, and transgenics, a few years from now, are the new hopes for undermining the resistance of the Aedes, an old acquaintance in Brazil. In the 1950’s, after an intense campaign to fight the mosquito, the Aedes was regarded as eradicated by international observers. The victory was short-lived.

In 1967, the Aedes was detected in Belém, probably brought in with tires smuggled from the Caribbean. Since then, it has proved to be very efficient in the capacity to adapt itself to the environment, assisted by the disposal without due care of tires, empty cans and plastic bottles, besides uncovered water tanks and plates beneath vases of plants, places favored as a breeding ground. No care is too much.

The Projects
Genetically modified mosquitoes: possible applications in the control of the transmission of malaria and dengue (00/12138-7); Modality: Young Researcher Program; Coordinator: Margareth de Lara Capurro  – USP; Investment: R$ 1,079,231.45 (Fapesp)
2. Development of alternatives methodologies in the control of mosquitoes of epidemiological importance: use of the RIDL method (05/55196-0); Modality: Young Researcher program; Coordinator: Mauro Toledo Marrelli – USP; Investment: R$438,627.17 (Fapesp)