An assay to detect filarial L3 in pools of mosquitoes requires 1) a ‘field-friendly’ method of collecting mosquitoes that preserves parasite RNA, 2) an effective RNA extraction method that isolates parasite RNA along with mosquito RNA, 3) identification of an L3-activated gene to ensure only infective stage parasites are detected, and 4) sensitive and species-specific detection of the L3-activated gene. The first two requirements, preservation and extraction of parasite RNA in mosquitoes, were successfully accomplished during our previous work on Brugia
. However, we were unable to identify a W. bancrofti
orthologue of the B. malayi
L3-activated diagnostic target (Accession # AA585578). This necessitated a search for a different L3-activated gene in W. bancrofti
Our strategy used bioinformatics to identify cuticle genes. Cuticle genes are known to have a heterochronic expression pattern in free-living nematode worms 
. In addition, a cuticle collagen gene was identified in our previous work as being L3-activated in B. malayi 
. In this study we identified eight W. bancrofti
collagens and three cuticlins and examined their expression profiles in detail using RNA isolated from mosquitoes at daily time points after feeding on infected blood. One of the eight collagen genes identified in this search (Accession # CK855340) was a gene that was previously identified by Vasuki et al 
(Accession # EU370160) as being a W. bancrofti
L3-diagnostic gene (col-2
). However, we found that W. bancrofti col-2
was expressed on day 6 PBM, prior to the first appearance of L3 on day 8 PBM in mosquitoes reared for this study. Thus, this gene was not L3-activated. Of the eleven cuticle-related targets investigated in this study, we identified three W. bancrofti
L3-activated genes including one collagen (Accession # CK855471) and two cuticlin genes (Accession # CK850637 and # AF125580).
Specificity of the primer/probe sets for the L3-activated targets was evaluated using B. malayi
, B. pahangi
, and D. immitis
infective mosquitoes due to availability of laboratory animal models for these species. There are other filarial parasites in endemic areas, but L3 stage material from these parasites was unavailable for testing. Nonetheless, given that the Wuchereria
species are very close evolutionary neighbors 
it is highly unlikely that a gene from a more distantly-related species would be more similar in sequence at the nucleotide level. W. bancrofti
specificity of both the cut-1.0
assays was confirmed, while the W. bancrofti
L3-activated collagen transcript (CK855471) was detected in mosquitoes harboring B. pahangi
L2 stage parasites making it unsuitable as a target for WbL3-detection in field samples. Although both cuticlins could be suitable targets for L3 detection, we selected cut-1.2
for the WbL3-detection assay due to the slightly later time point of earliest detection (day 9PBM versus day 8PBM).
One infective mosquito was detected in a pool of up to 30 mosquitoes using the multiplex real-time RT-PCR L3-detection assay with tph-1 and cut-1.2. As expected, the conventional assay is slightly less sensitive, detecting one infective mosquito in a pool of up to 20 mosquitoes. A potential limitation of this study is that the WbL3 assay was not tested at the level of single worm detection due to the difficulty in obtaining isolated W. bancrofti L3 parasites preserved for RNA extraction (fast-frozen in liquid nitrogen). It is not possible to preserve single worms in RNAlater solution because the high salt content does not allow the separation of the worm from the solution for RNA extraction. Thus, the sensitivity of the WbL3-detection assay can only be stated as per infective mosquito, not per L3 parasite.
With methods previously used for B. malayi, we multiplexed the WbL3-detection target with the constitutively expressed control gene tph-1 to enable simultaneous ‘any-stage’ detection in a standard RT-PCR assay. This allows both xenomonitoring and transmission risk to be evaluated in one test. It is important to note that the tph-1 assay detects an expression signal from both Brugia and Wuchereria, but not from the related zoonotic parasite D. immitis.
One consideration for the implementation of any new diagnostic technique is the practicality of using it as a monitoring or surveillance tool in the field. The storage of vectors in RNAlater eliminates any major limitations regarding mosquito collection. The mosquitoes can be stored for at least one day at ambient temperature and for several months to even years at −20°C or −80°C. Any laboratory that is already performing PCR would be able to use the conventional RT-PCR assays with no additional equipment investment. For the real-time assay, the investment of a real-time PCR instrument would be necessary in laboratories that do not already have such an instrument. The advantages to the real-time assay include a higher throughput level (reduced labor investment), increased sensitivity, as well as a reduction in potential contamination due to the elimination of post-PCR product handling. The real-time assay is a more cost efficient test and it is the preferred test to use if the equipment is available. Studies are currently underway to validate this new diagnostic tool for use in field-caught mosquitoes.
Over the past few decades much progress has been made in advancing diagnosis of LF but not in monitoring transmission. GPELF currently uses indirect human measures to evaluate the success of its primary goal, the interruption of transmission. An L3-detection assay provides a more direct measure of transmission risk and may be useful as a sensitive and non-invasive method for monitoring GPELF programs. This multiplex L3/‘any-stage’ detection assay could also be a non-invasive surveillance tool for early detection of LF resurgence following suspension of MDA by detecting both Mf in the community and potential transmission risk. L3 detection may also be useful for identifying mosquito species that are LF vectors in areas where this is not already known; non-vector mosquitoes should not harbor L3. Finally, this new tool may also be used to answer research questions such as the seasonality of transmission or the effect of MDA on transmission rates.