title: High throughput multiplex assay for species identification of Papua New Guinea malaria vectors: members of the Anopheles punctulatus (Diptera: Culicidae) species group.
abstract: Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 2], An. torresiensis [An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species-specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An. punctulatus, An. koliensis, An. farauti 1, An. hinesorum, and An. farauti 4). A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay. Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus, 85.3% for An. koliensis, and 88.9% for An. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 4. Misclassification results from variable coloration of the proboscis and overlap of An. punctulatus, An. koliensis, the An. farauti 4. This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.
authors.affiliation: Center for Global Health and Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. cnh@case.edu
authors.forename: Cara N
authors.initials: CN
authors.lastname: Henry-Halldin
authors.forename: Lisa
authors.initials: L
authors.lastname: Reimer
authors.forename: Edward
authors.initials: E
authors.lastname: Thomsen
authors.forename: Gussy
authors.initials: G
authors.lastname: Koimbu
authors.forename: Allison
authors.initials: A
authors.lastname: Zimmerman
authors.forename: John B
authors.initials: JB
authors.lastname: Keven
authors.forename: Henry
authors.initials: H
authors.lastname: Dagoro
authors.forename: Manuel W
authors.initials: MW
authors.lastname: Hetzel
authors.forename: Ivo
authors.initials: I
authors.lastname: Mueller
authors.forename: Peter
authors.initials: P
authors.lastname: Siba
authors.forename: Peter A
authors.initials: PA
authors.lastname: Zimmerman
mesh_terms: D000818:Animals; D000852:Anopheles / Q000145:classification* / Q000235:genetics; D001483:Base Sequence; D021901:DNA, Intergenic / Q000235:genetics; D017753:Ecosystem; D006801:Humans; D007303:Insect Vectors / Q000145:classification* / Q000235:genetics; D008288:Malaria / Q000635:transmission*; D008969:Molecular Sequence Data; D010219:Papua New Guinea; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D021901:DNA, Intergenic