A LAMP-based colorimetric assay to expedite field surveillance of the invasive mosquito species Aedes aegypti and Aedes albopictus

Background Yellow fever, dengue, chikungunya and Zika viruses are responsible for considerable morbidity and mortality in humans. Aedes aegypti and Aedes albopictus are the most important mosquito vectors involved in their transmission. Accurate identification of these species is essential for the implementation of control programs to limit arbovirus transmission, during suspected detections at ports of first entry, to delimit incursions or during presence/absence surveillance programs in regions vulnerable to invasion. We developed and evaluated simple and rapid colorimetric isothermal tests to detect these two mosquito species based on loop-mediated isothermal amplification (LAMP) targeting the ribosomal RNA internal transcribed spacer 1 (ITS1). Methodology/Principal findings Samples were prepared by homogenizing and heating at 99 oC for 10 min before an aliquot was added to the LAMP reaction. After 40 min incubation at 65 oC, a colour change indicated a positive result. The tests were 100% sensitive and species-specific, and demonstrated a limit of detection comparable with PCR-based detection (TaqMan chemistry). The LAMP assays were able to detect target species for various life stages tested (adult, 1st instar larva, 4th instar larva and pupa), and body components, such as legs, wings and pupal exuviae. Importantly, the LAMP assays could detect Ae. aegypti DNA in mosquitoes stored in Biogents Sentinel traps deployed in the field for 14 d. A single 1st instar Ae. aegypti larva could also be detected in a pool of 1,000 non-target 1st instar Aedes notoscriptus, thus expediting processing of ovitrap collections obtained during presence/absence surveys. A simple syringe-sponge protocol facilitated the concentration and collection of larvae from the ovitrap water post-hatch. Conclusions/Significance We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts. The LAMP assays described herein are useful adjuncts to laboratory diagnostic testing or could be employed as standalone tests. Their speed, ease-of-use, low cost and need for minimal equipment and training make the LAMP assays ideal for adoption in low-resource settings without the need to access diagnostic laboratory services.
Author summary Two species of mosquitoes, Aedes aegypti and Aedes albopictus, are important mosquito vectors that transmit yellow fever, dengue, chikungunya and Zika viruses. There are limited vaccine and antiviral options to treat these diseases, and so vector control (chemical or biological) is the main strategy for preventing virus transmission. Active mosquito surveillance employing a range of methods is critical for guiding control programs. The context where surveillance is undertaken includes assessing population dynamics in areas where Ae. aegypti and Ae. albopictus are established and transmitting viruses; delimiting their range during incursions; detecting importations at first ports of entry; or conducting broad-scale presence/absence surveillance in regions vulnerable to invasion. Effective surveillance is underpinned by accurate and rapid identification of collected specimens. Molecular methods can complement more traditional morphological identification in instances when sample numbers are large, specimens are damaged, when related species share overlapping morphology or when diagnostic features in early instar larvae are not developed. Laboratory-based DNA tests are available; however, these require sophisticated equipment and incur lag time for the sample to be shipped and tested, which results in critical delay to implement rapid intervention. In this work, we have developed simple rapid tests to detect Ae. aegypti and Ae. albopictus that can be performed at or near the field sites, require only a single reaction temperature, and which give a visual colour change when a positive sample is detected. The assays identified all life stages and body components (legs and wings) tested, as well as adult specimens that were held in traps under field conditions for two weeks. They also detected a single 1st instar larva in a sample of >1,000 non-target mosquitoes to expedite ovitrap processing and subsequent morphological identification. The assays should be a useful adjunct to laboratory testing or could be employed as standalone tests.