Absence of knockdown mutations in pyrethroid and DDT resistant populations of the main malaria vectors in Colombia.

BACKGROUND: Knockdown resistance (kdr) is a well-characterized target-site insecticide resistance mechanism that is associated with DDT and pyrethroid resistance. Even though insecticide resistance to pyrethroids and DDT have been reported in Anopheles albimanus, Anopheles benarrochi sensu lato (s.l.), Anopheles darlingi, Anopheles nuneztovari s.l., and Anopheles pseudopunctipennis s.l. malaria vectors in Latin America, there is a knowledge gap on the role that kdr resistance mechanisms play in this resistance. The aim of this study was to establish the role that kdr mechanisms play in pyrethroid and DDT resistance in the main malaria vectors in Colombia, in addition to previously reported metabolic resistance mechanisms, such as mixed function oxidases (MFO) and nonspecific esterases (NSE) enzyme families. METHODS: Surviving (n = 62) and dead (n = 67) An. nuneztovari s.l., An. darlingi and An. albimanus mosquitoes exposed to diagnostic concentrations of DDT and pyrethroid insecticides were used to amplify and sequence a ~ 225 bp fragment of the voltage-gated sodium channels (VGSC) gene. This fragment spanning codons 1010, 1013 and 1014 at the S6 segment of domain II to identify point mutations, which have been associated with insecticide resistance in different species of Anopheles malaria vectors. RESULTS: No kdr mutations were detected in the coding sequence of this fragment in 129 samples, 62 surviving mosquitoes and 67 dead mosquitoes, of An. darlingi, An. nuneztovari s.l. and An. albimanus. CONCLUSION: Mutations in the VGSC gene, most frequently reported in other species of the genus Anopheles resistant to pyrethroid and DDT, are not associated with the low-intensity resistance detected to these insecticides in some populations of the main malaria vectors in Colombia. These results suggest that metabolic resistance mechanisms previously reported in these populations might be responsible for the resistance observed.

Insecticide Resistance and Its Intensity in Populations of Malaria Vectors in Colombia.

Insecticide resistance in malaria vectors threatens malaria prevention and control efforts. In Colombia the three primary vectors, Anopheles darlingi, An. nuneztovari s.l., and An. albimanus, have reported insecticide resistance to pyrethroids, organophosphates, carbamates, and DDT; however, the insecticide resistance monitoring is not continuous, and the data on the prevalence of resistance is scarce and geographically limited. We describe the resistance levels and intensity of previously detected resistant populations among primary malaria vectors from the most endemic malaria areas in Colombia. The study was carried out in 10 localities of five states in Colombia. Bioassays were carried out following the methodology of CDC Bottle Bioassay using the discriminating concentration and in order to quantify the intensity the specimens were exposed to 2, 5, and 10X discriminating concentrations. Five insecticides were tested: deltamethrin, lambda-cyhalothrin, alpha-cypermethrin, permethrin, and DDT. The results provide evidence of low resistance intensity and resistance highly localized to pyrethroids and DDT in key malaria vectors in Colombia. This may not pose a threat to malaria control yet but frequent monitoring is needed to follow the evolution of insecticide resistance.

Spatial distributions of Anopheles species in relation to malaria incidence at 70 localities in the highly endemic Northwest and South Pacific coast regions of Colombia.

BACKGROUND: A proper identification of malaria vectors is essential for any attempt to control this disease. Between 40 and 47 Anopheles species have been recorded in Colombia, and eight species complexes have been identified in the last decade. An update of Anopheles species distribution and its relationship with malaria is required, particularly for newly identified members of species complexes. METHODS: A cross-sectional entomological study was conducted at 70 localities in the highest malaria transmission areas in Colombia. In each locality, immature and adult mosquitoes were collected. All specimens were determined using morphological characters and confirmed used restriction profiles of Internal Transcribed Spacer 2 (PCR-RFLP-ITS2), and Cytochrome c Oxidase I (COI) sequence gene. To detect natural Plasmodium infections, enzyme-linked immunosorbent assay and nested PCR analysis were used. Distribution of Anopheles species was spatially associated with malaria incidence. RESULTS: A total of 1736 larvae and 12,052 adult mosquitoes were determined in the 70 localities. Thirteen Anopheles species were identified. COI sequence analysis suggested 4 new lineages for Colombia: for Anopheles albimanus (An. albimanus B), Anopheles pseudopunctipennis s.l., Anopheles neivai (An. neivai nr. neivai 4), and Anopheles apicimacula. Two members of species complexes were identified, as: Anopheles nuneztovari C, and Anopheles albitarsis I. Another seven species were confirmed. Four mosquitoes were infected with Plasmodium species, An. albimanus B and An. nuneztovari C. In Northwest of Colombia, An. nuneztovari C, An. albimanus, and Anopheles darlingi were present in the municipalities with highest annual parasitic index (API) (>35 cases/1000 inhabitants). In the north of South Pacific coast, with a similar API, An. nuneztovari C were widely distributed inland, and the main species in coastal regions were An. albimanus B and An. neivai s.l. In the South Pacific coast bordering with Ecuador, 3 Anopheles species were found in municipalities with high API (15-88 cases/1000 inhabitants): An. albimanus B, Anopheles calderoni and An. neivai s.l. CONCLUSIONS: In the highest malaria areas of Colombia, 13 Anopheles species and four new lineages were found, which highlights the need for updating the species distribution. A DNA barcode analysis allowed the taxonomic identification to be refined, particularly for species complexes, and to improve the further understanding of their relation with malaria transmission.

Larval habitat characteristics of the main malaria vectors in the most endemic regions of Colombia: potential implications for larval control.

BACKGROUND: Malaria incidence has recently decreased globally and, as malaria elimination is envisioned as a possibility by the health authorities, guidance is needed to strengthen malaria control strategies. Larval source treatment, which could complement routine vector control strategies, requires knowledge regarding the Anopheles larval habitats. METHODS: A cross-sectional study was conducted in three of the most malaria-endemic regions in Colombia. A total of 1116 potential larval habitats in 70 villages were sampled in three states located in western Colombia: Cordoba, Valle del Cauca and Nariño. RESULTS: Overall, 17.5 % (195) of the potential larval habitats were found positive for different Anopheles species. A total of 1683 larvae were identified belonging to seven species: Anopheles albimanus, Anopheles calderoni, Anopheles darlingi, Anopheles neomaculipalpus, Anopheles nuneztovari s.l., Anopheles pseudopunctipennis, and Anopheles triannulatus. The most widely distributed species was An. nuneztovari s.l., which was found mainly in human-made fishponds in Cordoba and temporary puddles in Valle del Cauca. Anopheles albimanus and An. calderoni were associated with human-made wells or excavation sites in Nariño. Cordoba displayed the greatest Anopheles species diversity with a total of six species (Shannon diversity index H': 1.063). Although Valle del Cauca had four species, one more than Nariño, the diversity was lower because only one species predominated, An. nuneztovari s.l. The larval habitats with the highest Shannon diversity index were lagoons (H': 1.079) and fishponds (H': 1.009) in Cordoba, excavation sites in Nariño (H': 0.620) and puddles in Valle del Cauca (H': 0.764). CONCLUSIONS: This study provides important information regarding the larval habitats of the main malaria vectors in the most malaria-endemic regions of Colombia, which will be useful in guiding larval control operations.

[Insecticide susceptibility evaluation in Aedes aegypti populations of Caldas, Colombia, in 2007 and 2011]. / Evaluación de la sensibilidad a insecticidas en poblaciones de Aedes aegypti (Diptera: Culicidae) del departamento de Caldas, Colombia, en 2007 y 2011.

INTRODUCTION: Continuous use of insecticides for the control of dengue transmission may lead to decreased susceptibility levels in mosquito vector populations. Timely monitoring is necessary to ensure detection of any potential resistance problems. OBJECTIVE: To determine the susceptibility status of Aedes aegypti to insecticides used in public health in Caldas, Colombia, during 2007 and 2011. MATERIALS AND METHODS: Susceptibility tests to the organophosphates temephos, malathion, fenitrothion and pirimiphos methyl, as well as to the pyrethroid deltamethrin, were carried out using standard World Health Organization and Centers for Disease Control and Prevention protocols. RESULTS: In 2007, resistance to temephos was detected in Ae. aegypti populations from La Dorada with resistance ratios of 11.5 and 13.3, prompting the Caldas Health Department to suspend the use of this larvicide. A reduction in resistance ratios to temephos was observed in 2011, as well as an apparent resistance to pirimiphos methyl. All Ae. aegypti populations tested were susceptible to deltamethrin, malathion and fenitrothion in both years. CONCLUSION: Evaluating the resistance ratios during two distinct periods allowed a decrease in resistance to be detected after suspension of the use of temephos. Surveillance of mosquito populations for changes in susceptibility levels to the insecticides used in dengue control programs is therefore recommended.

Human biting activity, spatial-temporal distribution and malaria vector role of Anopheles calderoni in the southwest of Colombia.

BACKGROUND: Anopheles calderoni was first recognized in Colombia in 2010 as this species had been misidentified as Anopheles punctimacula due to morphological similarities. An. calderoni is considered a malaria vector in Peru and has been found naturally infected with Plasmodium falciparum in Colombia. However, its biting behaviour, population dynamics and epidemiological importance have not been well described for Colombia. METHODS: To assess the contribution of An. calderoni to malaria transmission and its human biting behaviour and spatial/temporal distribution in the southwest of Colombia, human landing catches (HLC) and larval collections were carried out in a cross-sectional, entomological study in 22 localities between 2011 and 2012, and a longitudinal study was performed in the Boca de Prieta locality in Olaya Herrera municipality between July 2012 and June 2013. All mosquitoes determined as An. calderoni were tested by ELISA to establish infection with Plasmodium spp. RESULTS: Larvae of An. calderoni were found in four localities in 12 out of 244 breeding sites inspected. An. calderoni adults were collected in 14 out of 22 localities during the cross-sectional study and represented 41.3% (459 of 1,111) of the collected adult specimens. Other species found were Anopheles albimanus (54.7%), Anopheles apicimacula (2.1%), Anopheles neivai (1.7%), and Anopheles argyritarsis (0.2%). In the localities that reported the highest malaria Annual Parasite Index (>10/1,000 inhabitants) during the year of sampling, An. calderoni was the predominant species (>90% of the specimens collected). In the longitudinal study, 1,528 An. calderoni were collected by HLC with highest biting rates in February, May and June 2013, periods of high precipitation. In general, the species showed a preference to bite outdoors (p < 0.001). In Boca de Prieta, two specimens of An. calderoni were ELISA positive for Plasmodium circumsporozoite protein: one for P. falciparum and one for Plasmodium vivax VK-210. This represents an overall sporozoite rate of 0.1% and an annual entomological inoculation rate of 2.84 infective bites/human/year. CONCLUSIONS: This study shows that An. calderoni is a primary malaria vector in the southwest of Colombia. Its observed preference for outdoor biting is a major challenge for malaria control.

Evaluación de la sensibilidad a insecticidas en poblaciones de Aedes aegypti (Diptera: Culicidae) del departamento de Caldas, Colombia, en 2007 y 2011 / Insecticide susceptibility evaluation in Aedes aegypti populations of Caldas, Colombia, in 2007 and 2011

Introducción. El uso continuo de insecticidas para el control de la transmisión del dengue puede generar una reducción en los niveles de sensibilidad de las poblaciones de los mosquitos vectores. La vigilancia oportuna es necesaria para detectar cualquier problema potencial de resistencia. Objetivo. Determinar el estado de la sensibilidad a insecticidas de uso en salud pública en poblaciones de Aedes aegypti del departamento de Caldas, Colombia, en el 2007 y el 2011. Materiales y métodos. Se realizaron pruebas de sensibilidad a los organofosforados temefós, malatión, fenitrotión y metil-pirimifós y al piretroide deltametrina, siguiendo las metodologías estandarizadas de la Organización Mundial de la Salud y de los Centers for Disease Control and Prevention de los Estados Unidos. Resultados. En el 2007 se encontró resistencia al temefós en dos barrios del municipio de La Dorada, con grados de resistencia de 11,5 y 13,3. Por ello, la Dirección Territorial de Salud de Caldas suspendió el uso de este larvicida en dichos barrios. Cuatro años después, en el 2011, se observó una reducción en los grados de resistencia al temefós, así como resistencia al metil-pirimifós. Todas las poblaciones evaluadas fueron sensibles a los insecticidas deltametrina, malatión y fenitrotión en los dos períodos. Conclusión. La cuantificación de los grados de resistencia en dos períodos permitió evidenciar la reducción de la resistencia al temefós después de suspender su uso. Se recomienda mantener la vigilancia de los niveles de sensibilidad de los insecticidas usados en los programas de control de dengue.

Introduction: Continuous use of insecticides for the control of dengue transmission may lead to decreased susceptibility levels in mosquito vector populations. Timely monitoring is necessary to ensure detection of any potential resistance problems. Objective: To determine the susceptibility status of Aedes aegypti to insecticides used in public health in Caldas, Colombia, during 2007 and 2011. Materials and methods: Susceptibility tests to the organophosphates temephos, malathion, fenitrothion and pirimiphos methyl, as well as to the pyrethroid deltamethrin, were carried out using standard World Health Organization and Centers for Disease Control and Prevention protocols. Results: In 2007, resistance to temephos was detected in Ae. aegypti populations from La Dorada with resistance ratios of 11.5 and 13.3, prompting the Caldas Health Department to suspend the use of this larvicide. A reduction in resistance ratios to temephos was observed in 2011, as well as an apparent resistance to pirimiphos methyl. All Ae. aegypti populations tested were susceptible to deltamethrin, malathion and fenitrothion in both years. Conclusion: Evaluating the resistance ratios during two distinct periods allowed a decrease in resistance to be detected after suspension of the use of temephos. Surveillance of mosquito populations for changes in susceptibility levels to the insecticides used in dengue control programs is therefore recommended.

Anopheles species composition explains differences in Plasmodium transmission in La Guajira, northern Colombia.

Malaria in La Guajira, the most northern state of Colombia, shows two different epidemiological patterns. Malaria is endemic in the municipality of Dibulla whereas in Riohacha it is characterised by sporadic outbreaks. This study aimed to establish whether differences in transmission patterns could be attributed to different vector species. The most abundant adult female species were Anopheles aquasalis, exclusive to Riohacha, and Anopheles darlingi, restricted to Dibulla. Anopheles mosquitoes were identified using morphology and the molecular markers internal transcribed spacer 2 and cytochrome c oxidase I. All specimens (n = 1,393) were tested by ELISA to determine natural infection rates with Plasmodium falciparum and Plasmodium vivax. An. darlingi was positive for P. vivax 210, with an infection rate of 0.355% and an entomological inoculation rate of 15.87 infective bites/person/year. Anopheles albimanus larvae were the most common species in Riohacha, found in temporary swamps; in contrast, in Dibulla An. darlingi were detected mainly in permanent streams. Distinctive species composition and larval habitats in each municipality may explain the differences in Plasmodium transmission and suggest different local strategies should be used for vector control.

Anopheles species composition explains differences in Plasmodium transmission in La Guajira, northern Colombia

Malaria in La Guajira, the most northern state of Colombia, shows two different epidemiological patterns. Malaria is endemic in the municipality of Dibulla whereas in Riohacha it is characterised by sporadic outbreaks. This study aimed to establish whether differences in transmission patterns could be attributed to different vector species. The most abundant adult female species were Anopheles aquasalis, exclusive to Riohacha, and Anopheles darlingi, restricted to Dibulla. Anopheles mosquitoes were identified using morphology and the molecular markers internal transcribed spacer 2 and cytochrome c oxidase I. All specimens (n = 1,393) were tested by ELISA to determine natural infection rates with Plasmodium falciparum and Plasmodium vivax. An. darlingi was positive for P. vivax 210, with an infection rate of 0.355% and an entomological inoculation rate of 15.87 infective bites/person/year. Anopheles albimanus larvae were the most common species in Riohacha, found in temporary swamps; in contrast, in Dibulla An. darlingi were detected mainly in permanent streams. Distinctive species composition and larval habitats in each municipality may explain the differences in Plasmodium transmission and suggest different local strategies should be used for vector control.

[Anopheles species present in the department of Putumayo and their natural infectivity with Plasmodium]. / Especies de Anopheles presentes en el departamento del Putumayo y su infección natural con Plasmodium.

INTRODUCTION: Putumayo is considered an endemic region for malaria transmission, mainly due to Plasmodium vivax. The vectors in this region are Anopheles darlingi , which has been found only in the municipality of Puerto Leguízamo, and An. rangeli and An. oswaldoi s.l. , which were recently incriminated as vectors in Puerto Asís. OBJECTIVE: The purpose of this study was to determine the role of An. benarrochi B in malaria transmission in Putumayo, given that it is the most abundant species biting humans. MATERIALS AND METHODS: Collections of immature and adult stages of Anopheles spp. were made between 2006 and 2008 in the municipalities of Puerto Leguízamo and Puerto Asís in Putumayo, and sequences of internal transcribed spacer 2 ( ITS-2 ) of ribosomal DNA and the mitochondrial gene COI were obtained to confirm the morphological determinations. ELISA was carried out for P. vivax and P. falciparum infectivity. RESULTS: A total of 6,238 specimens were identified, distributed in 11 species: An. albitarsis s.l. (1.83%), An. benarrochi B (72.35%), An. braziliensis (0.05%), An. costai (0.06%), An. darlingi (19.37%), An. mattogrossensis (0.08%), An. neomaculipalpus (0.13%), An. oswaldoi s.l. (0.64%), An. punctimacula (0.03%), An. rangeli (5.12%), and An. triannulatus s.l. (0.34%). A total of 5,038 adults were assessed by ELISA and 5 were found positive for P. vivax 210 and VK 247, all belonging to An. benarrochi B. CONCLUSION: The results suggest that An. benarrochi B plays a role in the transmission of P. vivax in Putumayo due to its high human contact and natural infection with Plasmodium sp.

Especies de Anopheles presentes en el departamento del Putumayo y su infección natural con Plasmodium / Anopheles species present in the department of Putumayo and their natural infectivity with Plasmodium

Introducción. El departamento del Putumayo es una región endémica para malaria, o paludismo, causada principalmente por Plasmodium vivax . Los vectores en esta región incluyen Anopheles darlingi , el cual se ha encontrado solamente en el municipio de Puerto Leguízamo, y recientemente se incriminaron como vectores en Puerto Asís a las especies An. rangeli y An. oswaldoi . Objetivo. El propósito del trabajo fue determinar el papel de An. benarrochi B en la transmisión de malaria en este departamento, ya que se reporta como la especie más abundante que pica a los humanos. Materiales y métodos. Se recolectaron larvas y adultos de Anopheles spp. entre el 2006 y el 2008 en los municipios Puerto Leguízamo y Puerto Asís, y se obtuvieron secuencias del gen ITS-2 y del gen mitocondrial COI para confirmar las determinaciones taxonómicas por morfología. Se practicó la prueba ELISA para establecer la infección por P. vivax y P. falciparum. Resultados. Se identificaron 6.238 individuos correspondientes a 11 especies: An. albitarsis s.l. (1,83 %), An. benarrochi B (72,35 %), An. braziliensis (0,05 %), An. costai (0,06 %), An. darlingi (19,37 %), An. mattogrossensis (0,08 %), An. neomaculipalpus (0,13 %), An. oswaldoi s.l. (0,64 %), An. punctimacula (0,03 %), An. rangeli (5,12 %) y An. triannulatus s.l. (0,34 %). Se evaluaron 5.038 adultos por ELISA y 5 se encontraron positivos para P. vivax 210 y VK 247, todos pertenecientes a la especie An. benarrochi B. Conclusión. Los resultados sugieren que An. benarrochi B juega un papel en la transmisión de P. vivax en el departamento de Putumayo, dada su alta atracción por los humanos y su infección natural con Plasmodium spp.

Introduction: Putumayo is considered an endemic region for malaria transmission, mainly due to Plasmodium vivax. The vectors in this region are Anopheles darlingi , which has been found only in the municipality of Puerto Leguízamo, and An. rangeli and An. oswaldoi s.l. , which were recently incriminated as vectors in Puerto Asís. Objective: The purpose of this study was to determine the role of An. benarrochi B in malaria transmission in Putumayo, given that it is the most abundant species biting humans. Materials and methods: Collections of immature and adult stages of Anopheles spp. were made between 2006 and 2008 in the municipalities of Puerto Leguízamo and Puerto Asís in Putumayo, and sequences of internal transcribed spacer 2 ( ITS-2 ) of ribosomal DNA and the mitochondrial gene COI were obtained to confirm the morphological determinations. ELISA was carried out for P. vivax and P. falciparum infectivity. Results: A total of 6,238 specimens were identified, distributed in 11 species: An. albitarsis s.l. (1.83%), An. benarrochi B (72.35%), An. braziliensis (0.05%), An. costai (0.06%), An. darlingi (19.37%), An. mattogrossensis (0.08%), An. neomaculipalpus (0.13%), An. oswaldoi s.l. (0.64%), An. punctimacula (0.03%), An. rangeli (5.12%), and An. triannulatus s.l. (0.34%). A total of 5,038 adults were assessed by ELISA and 5 were found positive for P. vivax 210 and VK 247, all belonging to An. benarrochi B. Conclusion: The results suggest that An. benarrochi B plays a role in the transmission of P. vivax in Putumayo due to its high human contact and natural infection with Plasmodium sp.