Detection of Wuchereria bancrofti L3 larvae in mosquitoes: a reverse transcriptase PCR assay evaluating infection and infectivity

Abstract

Background: 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).

Methodology/principal findings: 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.

Conclusions/significance: 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.

Figure 1. Primer and probe alignment with cut-1.2 sequences of W. bancrofti and B. malayi.

A portion of the cut-1.2 sequences from B. malayi and W. bancrofti have been aligned with the primers and probes designed for the qRT-PCR cut-1.2 detection assay. The nucleotides in red (red arrows) represent single nucleotide polymorphisms between the Brugia and Wuchereria transcripts that provide the specificity of the target. The exons are differentiated in the W. bancrofti sequence by lower and upper case letters (vertical red bar). The probe spans the exon-exon boundary to prevent detection of any contaminating genomic DNA.

Figure 2. Conventional RT-PCR detection of W. bancrofti tph-1 and cut-1.2 in a mosquito time-course.

Time-course set C illustrates no amplification of the L3-activated cut-1.2 transcript (123 bp) in time-points prior to L3 development, while the control tph-1 transcript (153 bp) is detected in all time-points indicating that parasite RNA was present. Panel A shows mosquitoes collected from 0–8 days dPBM and panel B shows mosquitoes collected 9–13, and 16 dPBM. dPBM = the number of days post blood meal, Un Cxp = Unfed Cx. pipiens mosquitoes, Wb gDNA = W. bancrofti genomic DNA, NTC = No template control.

Figure 3. Sensitivity testing of the W. bancrofti multiplex L3-detection assay by conventional RT-PCR.

The tph-1 transcript (‘any-stage’ detection) is detected in all samples, while the cut-1.2 transcript (L3-detection) is only detected in samples of pool size up to 20 mosquitoes. The L3-detection sensitivity limit by conventional RT-PCR is one infective mosquito in a pool of 20 mosquitoes. 1∶10 = one bloodfed mosquito (day 16 post blood meal) in a pool of 10 mosquitoes, 1∶15 = one bloodfed mosquito in a pool of 15 mosquitoes, etc., 5∶10 = 5 bloodfed mosquitoes in a pool of 10 mosquitoes. Un Cxp = Unfed Cx. pipiens, NTC = no template control.