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Background  Accurate  Anopheles  species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the  Anopheles funestus  group multiplex PCR assay, it was noted that  Anopheles arabiensis  can be misidentified as  Anopheles leesoni , a zoophilic member of the  An. funestus  group. The aim of this project was, therefore, to ascertain whether other members of the  Anopheles gambiae  complex can also be misidentified as  An. leesoni  when using the standard  An. funestus  multiplex PCR.    Methods  The  An. funestus  multiplex PCR was used to amplify DNA from  An. gambiae  complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and  An. leesoni  species-specific primers (LEES), were sequence analysed. Additionally,  An. leesoni  DNA was processed through the diagnostic  An. gambiae  multiplex PCR to determine if this species can be misidentified as a member of the  An. gambiae  complex.    Results  Laboratory-colonized as well as field-collected samples of  An. arabiensis ,  An. gambiae ,  Anopheles merus ,  Anopheles quadriannulatus, Anopheles coluzzii  as well as  Anopheles moucheti  produced an amplicon of similar size to that of  An. leesoni  when using an  An. funestus  multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the  An. gambiae  complex and  An. moucheti . The reverse was not true, i.e. the  An. gambiae  multiplex PCR does not amplify DNA from  An. leesoni .    Conclusion  This investigation shows that  An. arabiensis ,  An. gambiae ,  An. merus ,  An. quadriannulatus, An. coluzzii  and  An. moucheti  can be misidentified as  An. leesoni  when using  An. funestus  multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as  An. leesoni , the  An. gambiae  multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

Member species of the Anopheles gambiae complex can be misidentified as Anopheles leesoni