title: Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies.
abstract: Without an adequate DNA extraction protocol, the identification of Plasmodium species in whole mosquitoes by PCR is difficult because of the presence of reaction inhibitors from the insects. In this study, eight DNA extraction protocols were tested, from which a chelex-based protocol was selected. Then a semi-nested multiplex PCR technique that detects and distinguishes among the four human Plasmodium species in single mosquitoes and in pools of up to 100 mosquitoes was optimized. The technique was used to detect P. vivax in wild-caught Anopheles pseudopunctipennis from a village in the Andean valleys of Bolivia in May 2003. The prevalence of infection was 0.9%. This is the first direct evidence of P. vivax transmission by this vector in this country. The extraction and PCR technique presented here can be useful to: (1) estimate Plasmodium prevalence in Anopheles populations in low prevalence areas where large numbers of individual mosquitoes would need to be processed to obtain a reliable estimate; (2) incriminate Anopheles species as malaria vectors; (3) identify all the circulating Plasmodium species in vectors from an area; (4) detect mixed infections in mosquitoes; and (5) detect mosquitoes with low-level parasite infections.
authors: Caractérisation et Contrôle des Populations de Vecteurs, Institut de Recherche pour le Développement (IRD), CP 9214, La Paz, Bolivia. lardeux@ird.fr
authors: Frédéric
authors: F
authors: Lardeux
authors: Rosenka
authors: R
authors: Tejerina
authors: Claudia
authors: C
authors: Aliaga
authors: Raul
authors: R
authors: Ursic-Bedoya
authors: Carl
authors: C
authors: Lowenberger
authors: Tamara
authors: T
authors: Chavez
mesh_terms: D000818:Animals; D000852:Anopheles / Q000469:parasitology*; D001838:Bolivia / Q000453:epidemiology; D016054:DNA, Protozoan / Q000302:isolation & purification*; D006801:Humans; D016778:Malaria, Falciparum / Q000453:epidemiology / Q000469:parasitology*; D010961:Plasmodium / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D016054:DNA, Protozoan