Detection of Wuchereria bancrofti L3 Larvae in Mosquitoes: A Reverse Transcriptase PCR Assay Evaluating Infection and Infectivity

Abstract

Detection of filarial DNA in mosquitoes by PCR cannot differentiate infective mosquitoes from infected mosquitoes. In order to evaluate transmission risk an assay is needed that can specifically detect infective L3 stage parasites. We now report the development of an assay that specifically detects the infective stage of Wuchereria bancrofti in mosquitoes. The assay detects an L3-activated mRNA transcript by reverse-transcriptase PCR (RT-PCR). W. bancrofti cuticle-related genes were selected using bioinformatics and screened as potential diagnostic target genes for L3 detection in mosquitoes. Expression profiles were determined using RT-PCR on RNA isolated from mosquitoes collected daily across a two-week period after feeding on infected blood. Conventional multiplex RT-PCR and real-time multiplex RT-PCR assays were developed using an L3-activated cuticlin transcript for L3 detection and a constitutively expressed transcript, tph-1, for ‘any-stage’ detection. This assay can be used to simultaneously detect W. bancrofti infective stage larvae and ‘any-stage’ larvae in pooled vector mosquitoes. This test may be useful as a tool for assessing changes in transmission potential in the context of filariasis elimination programs. Lymphatic filariasis is a disabling and disfiguring disease caused by a parasite that is transmitted by a mosquito. The life cycle of the parasite requires two hosts: the mosquito vector and the human host. Part of the developmental life cycle of the parasite occurs in the mosquito and the other part in the human host. The parasite develops through four stages in the mosquito, only the last of which is infectious to humans. The third larval stage (L3) is the infective stage that initiates human infections when infective mosquitoes bite humans. There is currently a global program attempting to eliminate this disease by administering drugs to affected communities with the goal of interrupting transmission of the parasite. The new diagnostic tool described in this paper uses molecular techniques to specifically detect the infective stage of the parasite in mosquitoes. Many mosquitoes can be tested at one time to assess the risk of ongoing transmission of filariasis in communities. In addition, this new L3-detection assay can simultaneously detect whether the mosquitoes contain ‘any-stage’ of the parasite. This provides information on infection rates in humans in the community. Both pieces of information can be used in assessing the progress of disease elimination efforts.