| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In Brazil, public health programs since 1967 have used mostly organophosphates in the control of Ae. aegypti.4 However, this procedure has not prevented the appearance of several dengue epidemics; since 1986, the number of dengue cases has increased in several regions of this country.5,6
Apart from some isolated reports, mainly in the State of Sao Paulo,7,8 the insecticide resistance status of Ae. aegypti in Brazil has not been systematically verified. However, field personnel have recently reported a decrease in the persistence of temephos, the main larvicide used to control Ae. aegypti, suggesting that mosquito populations have acquired resistance to this organophosphate. For this reason, in 1999 the National Health Foundation (Fundação Nacional de Saúde [FUNASA]) instituted the first nationally integrated program to monitor the resistance status of this dengue vector to insecticides commonly used in all regions of Brazil. Several municipalities were chosen, based on the number of dengue cases or the mosquito infestation levels. In this program, the capitals of each state were included. Our laboratory has been responsible for 10 municipalities, seven in the state of Rio de Janeiro State and three in the state of Espírito Santo. Bioassays to detect resistance of larvae and adults was performed according to the methodologies recommended by the World Health Organization (WHO)9 and the Centers for Disease Control and prevention (CDC),10 respectively.
|
MATERIALS AND METHODS |
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The Rockefeller reference strain, which served as the susceptibility control in all assays, was maintained continuously in the laboratory.
|
60,000 buildings, 100 ovitraps; 60,000–120,000 buildings, 150 ovitraps; 120,000–500,000 buildings, 200 ovitraps; >500,000 buildings, 300 ovitraps.14 Field collection of eggs was done between August and September 1999. After five days, the ovitraps were collected and carried to the laboratory. The presence of eggs in each ovitrap was then scored and eggs from positive ovitraps were pooled according to their municipality. Laboratory generation of F1 mosquitoes from field populations. Positive ovitraps were immersed in dechlorinated water to induce larvae hatching. After 24 hours, larvae were transferred to rectangular basins containing two liters of dechlorinated water. Dog food (Purina, Paulinia, SP) was supplied daily to feed the larvae. Pupae were transferred to cylindrical cardboard cages (18 x 30 cm) and the resulting adults (Ae. aegypti or Ae. albopictus) were identified. Only Ae. aegypti mosquitoes were kept for further analysis. These were transferred to square metal cages (30 cm per side) and first fed on anesthetized guinea pigs three days after emergence of the adults. Three days after the blood meal, eggs were collected for three days in small plastic cups containing wet filter paper. Paper strips containing the eggs were then allowed to dry in an insectary and served as the source of F1 mosquitoes for the bioassays. Although blood feeding and collection of eggs had been performed weekly and repeated as many times as possible, some bioassays, due to insufficient amounts of F1 eggs, were performed with the F2 generation mosquitoes.
Larval bioassays. Temephos resistance bioassays were performed with F1 larvae, according to the WHO recommended procedure and parameters.9 The diagnostic dose (DD), which corresponded to twice the concentration of the 99% lethal dose (LD99) for the susceptible strain, was used. According to WHO standardization, the temephos DD for A. aegypti is 0.012 mg/L.9 Previous tests in the laboratory had confirmed a dose of 0.006mg/L as the LD99 for the Rockefeller strain.
Twelve samples were used in each test. Each sample consisted of 20 larvae in 250 mL: 8 samples with 0.012 mg of Temephos/L (1 mL of a 3 mg/L alcoholic solution) and four samples with 1 mL of ethanol. Mortality was scored 24 hours after the beginning of the test. Each bioassay was repeated three times for all municipalities.
To define the resistance ratio to Temephos, F2 larvae were subjected to different insecticide concentrations, following the procedure recommended by the WHO.9
Adult bioassays. Malathion, temephos, and fenitrothion resistance bioassays for adults were performed with F1 or F2 mosquitoes and followed the CDC methodology, based on the use of 250-mL glass bottles impregnated with insecticides.10 Our previous assays indicated that 400 µg of malathion per bottle, 900 µg of temephos per bottle, and 800 µg of fenithrotion per bottle were sufficient to induce 100% mortality in the susceptible Rockefeller strain. These conditions were similar to those previously defined for malathion and temephos.15
Each complete test consisted of four bottles, three impregnated with insecticide (1 mL of acetone containing 40, 90, and 80 µL of a 10 mg/mL stock solution made in acetone of malathion, temephos, and fenithrotion, respectively) and one with acetone (1 mL). Twenty non-blood-fed females, 3–5 days old, were used in each bottle. Mortality was scored at 15-minute intervals for 45 minutes for malathion and for 75 minutes for temephos and fenitrothion. In cases of resistance, the test was performed for 120 minutes. Each test was repeated three times for mosquitoes derived from all municipalities, except when indicated otherwise.
Evaluation criteria and statistical analysis. In all cases, the resistance/susceptible status of mosquito populations was evaluated according to WHO criteria.16 Mortality greater than 98% indicates susceptibility, mortality less than 80% defines resistance, and mortality between 80% and 98% is suggestive of an incipient altered susceptibility, indicating the need for surveillance of the corresponding population.
The results of dose response tests were subjected to probit analysis using the computer program Polo-PC (LeOra Software, Berkeley, CA) to define lethal concentrations and resistance ratios (RRs).
|
RESULTS |
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The Rockefeller (control) strain showed 100% mortality, thus corroborating its susceptibility to the insecticide and validating our results.
|
In the state of Rio de Janeiro, the situation was worse: the seven populations analyzed showed resistance, with mortality rates less than 80% (Table 1
). The degree of resistance seemed to be higher in the capital (Rio de Janeiro) and neighboring cities, where the level of mortality did not exceed 35%, except in São João de Meriti and Nova Iguaçu (44% and 58.2%, respectively). Campos dos Goytacazes, which is located in northern part of this state, had a mortality of 74%, a rate that indicates resistance but reveals a less drastic situation when compared with the more populated area in the state of Rio de Janeiro.
Larval bioassays: resistance ratio for temephos.
Larvae from three Aedes populations were subjected to temephos dose-response tests (Table 2). The results were consistent with those of the diagnostic dose bioassays, with the municipality showing the lowest mortality level (São Gonçalo = 23.5%; Table 1) having the highest RR (RR90 = 12.41; Table 2). The other two localities (São João de Meriti and Duque de Caxias) exhibited moderate levels of resistance. However, although the F1 generation has been used in the diagnostic-dose bioassays, the RR was determined with the F2 generation that was grown in the laboratory without any selective pressure. Thus, the RRs may be underestimated.
|
. When compared with the LD-P line from the susceptible Rockefeller reference strain, those of field populations are shifted to the right, indicating resistance. The shallow slope obtained for LD-P lines from field populations indicates their (expected) higher diversity when compared with the Rockefeller strain.17
|
) showed that for A. aegypti populations from the state of Espírito Santo, results obtained with temephos were consistent with those in larval bioassays (Table 1). Vila Velha showed resistance in both tests (mortality = 45.5% and 58.3%, respectively, in larva and adult assays), while mortality levels in Cariacica and Vitória indicated the need for surveillance.
|
). However, adult mortality data showed that only three populations in this state were resistant to temephos (Duque de Caxias = 79.3%; Rio de Janeiro = 11.7%, and São Gonçalo, = 74.3%; Table 3). Thus, since temephos was used as a larvicidae, different levels of resistance can be exhibited during development.
Our results show higher resistance to temephos in larvae, compared with that in adults, in 87.5% of the municipalities analyzed (Tables 1 and 3). However, the reverse was observed in the municipality of Rio de Janeiro (larval mortality = 32.3%, adult mortality = 11.7%). It has already been observed that the resistance pattern exhibited by larvae and adults of a given population relative to the same insecticide may differ.18 Monitoring temephos adult bioassays for 120 minutes indicated that 100% mortality was not observed in any population (with the exception of São João de Meriti), which corroborated the altered susceptibility status to this insecticide.
Although fenitrothion is also an organophosphate, adult bioassays with this insecticide showed that resistance was generally lower when compared with that to temephos. Two populations remained susceptible to this insecticide (Duque de Caxias and Nova Iguaçu, 100% mortality in 75 minutes), but true resistance (mortality less than 80%) was not observed. The results of these bioassays after 120 minutes showed 100% mortality in four of 10 populations and confirmed the need for surveillance in the other six (Table 3).
Resistance to malathion was also lower when compared with that to temephos. Bioassays with adult females showed an incipient susceptible altered status in the three municipalities. Monitoring the tests for 120 minutes resulted in 100% mortality only in the Vila Velha population (Table 3). None of the Ae. aegypti populations tested in the state of Rio de Janeiro was resistant to malathion. In fact, in four municipalities (Nova Iguaçu, Rio de Janeiro, São Gonçalo, and São João de Meriti), mosquitoes remained susceptible, while in the remaining three municipalities (Campos dos Goytacazes, Duque de Caxias, and Niterói), incipient alterations in susceptibility were observed. In these latter cases, monitoring for 120 minutes did not result in 100% mortality.
It is interesting to note that two malathion-susceptible populations (São Gonçalo and Rio de Janeiro) also showed the highest resistance levels to temephos. These populations also exhibited a moderate change in susceptibility to fenitrothion.
|
DISCUSSION |
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aedes albopictus has been found in the state of Espirito Santo since 1986,19 but because this mosquito has not been incriminated as a dengue vector in Brazil, no control action has been taken. Vector surveillance detected Ae. aegypti in this state for the first time in 1993 in the municipality of Vila Velha. In 1994, Ae. aegypti was detected in other municipalities in the state of Espírito Santo, including Cariacica and Vitória. In 1995, mosquito infestation and dengue transmission were confirmed in the state.5 The application of insecticides (mainly temephos and malathion) started in 1993 in Vila Velha and in 1994 in the other municipalities. It is important to note that Vila Velha is infested with Culex quinquefasciatus, and has been subjected to local application of different insecticides during the 1990s. Vila Velha has also been subjected to periodically exposures to DDT during episodes of malaria transmission, the last one occurring in 1992.
The data presented here are part of the first integrated effort in Brazil of monitoring Ae. aegypti insecticide resistance. This effort was derived from the observation that the dengue vector is spreading in the country despite control efforts. According to FUNASA, 1,752 municipalities were infested with Ae. aegypti in 1995, and this number increased to 3,535 in 1999. Also, the number of dengue cases is not encouraging: 137,308 cases were reported in 1995, and this increased to 559,237 in 1998. This increase was due mainly to cases reported in the southeastern region of Brazil, the most populated area in the country (46,845 cases in 1995 and 250,303 cases in 1998). Although massive attempts to reduce dengue were undertaken in 1998 through the extensive use of chemical insecticides, 209,294 cases were reported in 1999, a number that is still far from desirable.23 The persistent increase in the number of Aedes-infested municipalities, together with the appearance of several dengue epidemics in urban areas, emphasizes the ineffectiveness of classic control methods and the need to monitor resistance in Brazil.
Our results show true or incipient resistance to at least one of the three organophosphates tested in all populations investigated in the states of Rio de Janeiro and Espírito Santo. This is consistent with the intense use of organophosphates in the country since 1986. A positive correlation between the intensity of insecticide use and the resistance level was observed in both states. In Rio de Janeiro, municipalities located in the metropolitan region showed higher resistance levels than in Campos dos Goytacazes, which is located in the northern part of this state. In Espírito Santo, true resistance to temephos was observed only in Vila Velha, a municipality that has been historically exposed to a high insecticide selective pressure.
Our data indicate the need of adopting a vector control strategy structured on a case-by-case basis. Since 2001, the alternative Bacillus thuringiensis insecticide (Bti) has been used to control Ae. aegypti in all municipalities that showed resistance to temephos. We are now investigating the biochemical nature of resistance in all concerned populations to provide more information to the Brazilian dengue control program. Additional biologic assays are also planned to analyze the evolution of insecticide resistance in various populations.
Received December 3, 2001. Accepted for publication September 30, 2002.
Acknowledgments: We thank the Núcleo de Entomologia do Rio de Janeiro (NERJ/FUNASA) for collection of eggs and help with some of the biologic assays.
Financial support: This work was supported by Fundação Oswaldo Cruz (Papes II Program), Programa de Erradicação do Aedes aegypti (PEAa), Financiadora de Estudos e Projetos do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Nacional de Saúde (FUNASA), and the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR).
Authors’ addresses: José Bento Pereira Lima, Marcella Pereira DaCunha, Ima Aparecida Braga, and Denise Valle, Departamento de Entomologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, Brazil, CEP 21045-900, Telephone: 55-21-2580-6598, Fax: 55-21-2573-4468. Ronaldo Carneiro da Silva Júnior, Laboratório de Entomologia, Instituto de Biologia do Exército, Rua Francisco Manoel, 102, Benfica, Rio de Janeiro, RJ, Brazil, CEP 20911-270, Telephone: 55-21-2580-6598, Fax: 55-21-2573-4468. Allan Kardec Ribeiro Galardo and Sidinei da Silva Soares, Coordenação Regional da Fundação Nacional de Saúde do Rio de Janeiro, Rua Coelho e Castro, 6/7° Andar, Bairro Saúde, Rio de Janeiro, RJ, Brazil, CEP 20081-060, Telephone: 55-21-2263-8143, Fax: 55-21-2263-6754. Ricardo Pimentel Ramos, Coordenação Regional da Fundação Nacional de Saúde do Espírito Santo, Rua Moacir Straus, 85, Praia do Canto, Vitória, ES, Brazil, CEP 29055-630.
Reprint requests: Denise Valle, Departamento de Entomologia, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Pavilhão Carlos Chagas, 4° Andar, Av. Brasil, 4365, Manguinhos, Rio de Janeiro, RJ, Brazil, CEP 21045-900, E-mail: dvalle{at}ioc.fiocruz.br.
|
REFERENCES |
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Hirunkanokpun, J. O. Carlson, and P. Kittayapong Evaluation of Mosquito Densoviruses for Controlling Aedes aegypti (Diptera: Culicidae): Variation in Efficiency due to Virus Strain and Geographic Origin of Mosquitoes Am J Trop Med Hyg, May 1, 2008; 78(5): 784 - 790. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Montella, A. J. Martins, P. F. Viana-Medeiros, J. B. P. Lima, I. A. Braga, and D. Valle Insecticide Resistance Mechanisms of Brazilian Aedes aegypti Populations from 2001 to 2004 Am J Trop Med Hyg, September 1, 2007; 77(3): 467 - 477. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
