Abstract
BackgroundYellow 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 findingsSamples 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/SignificanceWe 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.
Highlights
Arboviruses are arthropod-borne viruses that occur worldwide and cause a large burden of human mortality and morbidity
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)
We describe the development of LAMP assays for species identification and demonstrate their direct application for surveillance in different field contexts
Summary
Arboviruses are arthropod-borne viruses that occur worldwide and cause a large burden of human mortality and morbidity. Some of the most important arboviruses are yellow fever (YFV), dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) viruses, all of which are transmitted primarily by the invasive and urbanized mosquito species, Aedes aegypti and Aedes albopictus [1]. Ae. albopictus (the “Asian tiger mosquito”) originated in Southeast Asia and has spread more recently to become an important arbovirus vector in infested regions. It has colonized parts of Africa, North and South America, Europe and the islands of the Western Pacific [4], with much of its dispersal linked to the used tire trade [5]. Statistical models of spatial spread predict that both species are likely to continue to expand their geographical distributions [8]
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