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Rapid protein profiling facilitates surveillance of invasive mosquito species.

ABSTRACT: BACKGROUND: Invasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for easy and rapid species identification. METHODS: Individual protein profiles were generated using five eggs each of nine aedine species (Ae. aegypti, Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) from various geographical origins, and species-specific biomarker mass sets could be generated. A blinded validation using our reference data base for automated egg identification was performed. In addition, pools of 10 aedine eggs (132 two-species and 18 three-species pools) in different ratios were evaluated. RESULTS: Specific biomarker mass sets comprising 18 marker masses could be generated for eggs of nine container-inhabiting aedine species, including all the major invasive and indigenous species of Europe and North America. Two additional masses shared by all investigated aedine species are used as internal calibrators. Identification of single eggs was highly accurate (100% specificity, 98.75% sensitivity), and this method is also of value for the identification of species in pools of ten eggs. When mixing two or three species, all were identified in all pools in at least 2 or 1 of the 4 loaded replicates, respectively, if the "lesser abundant" species in the pool accounted for three or more eggs. CONCLUSIONS: MALDI-TOF MS, which is widely applied for routine identification of microorganisms in clinical microbiology laboratories, is also suited for robust, low-cost and high throughput identification of mosquito vectors in surveillance programmes. This tool can further be developed to include a wide spectrum of arthropods but also other Metazoa for which surveillance is required, and might become the method of choice for their centralised identification via online platforms.

SUBMITTER: Schaffner F

PROVIDER: S-EPMC4022357 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Rapid protein profiling facilitates surveillance of invasive mosquito species.

Schaffner Francis F Kaufmann Christian C Pflüger Valentin V Mathis Alexander A

Parasites & vectors 20140331

<h4>Background</h4>Invasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evalu ...[more]

PMID: 24685094

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Project description:Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information, data can also be reviewed at any later stage for diagnostic or functional patterns without the need for re-designing and re-processing biological material.

Project description: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.

Project description:BackgroundMalaria impacts nearly 250 million individuals annually. Specifically, Uganda has one of the highest burdens, with 13 million cases and nearly 20,000 deaths. Controlling the spread of malaria relies on vector surveillance, a system where collected mosquitos are analyzed for vector species' density in rural areas to plan interventions accordingly. However, this relies on trained entomologists known as vector control officers (VCOs) who identify species via microscopy. The global shortage of entomologists and this time-intensive process cause significant reporting delays. VectorCam is a low-cost artificial intelligence-based tool that identifies a mosquito's species, sex, and abdomen status with a picture and sends these results electronically from surveillance sites to decision makers, thereby deskilling the process to village health teams (VHTs).ObjectiveThis study evaluates the usability of the VectorCam system among VHTs by assessing its efficiency, effectiveness, and satisfaction.MethodsThe VectorCam system has imaging hardware and a phone app designed to identify mosquito species. Two users are needed: (1) an imager to capture images of mosquitos using the app and (2) a loader to load and unload mosquitos from the hardware. Critical success tasks for both roles were identified, which VCOs used to train and certify VHTs. In the first testing phase (phase 1), a VCO and a VHT were paired to assume the role of an imager or a loader. Afterward, they swapped. In phase 2, two VHTs were paired, mimicking real use. The time taken to image each mosquito, critical errors, and System Usability Scale (SUS) scores were recorded for each participant.ResultsOverall, 14 male and 6 female VHT members aged 20 to 70 years were recruited, of which 12 (60%) participants had smartphone use experience. The average throughput values for phases 1 and 2 for the imager were 70 (SD 30.3) seconds and 56.1 (SD 22.9) seconds per mosquito, respectively, indicating a decrease in the length of time for imaging a tray of mosquitos. The loader's average throughput values for phases 1 and 2 were 50.0 and 55.7 seconds per mosquito, respectively, indicating a slight increase in time. In terms of effectiveness, the imager had 8% (6/80) critical errors and the loader had 13% (10/80) critical errors in phase 1. In phase 2, the imager (for VHT pairs) had 14% (11/80) critical errors and the loader (for VHT pairs) had 12% (19/160) critical errors. The average SUS score of the system was 70.25, indicating positive usability. A Kruskal-Wallis analysis demonstrated no significant difference in SUS (H value) scores between genders or users with and without smartphone use experience.ConclusionsVectorCam is a usable system for deskilling the in-field identification of mosquito specimens in rural Uganda. Upcoming design updates will address the concerns of users and observers.

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Rapid protein profiling facilitates surveillance of invasive mosquito species.

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Rapid protein profiling facilitates surveillance of invasive mosquito species.

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