@article{2b9c9e7ae5fd43d8b85936cce2d2d5bd,
title = "New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes",
abstract = "An essential component of malaria vector control programmes is the detection of Plasmodium falciparum within its mosquito vectors, particularly in the salivary glands where the infective sporozoites reside. Several protocols have been developed for this purpose; however they require dissection of mosquito specimens prior to analysis. Here, a novel one-step RT-qPCR TaqMan diagnostic assay was developed for mosquitoes with infective Plasmodium falciparum sporozoites in the salivary glands. It is based on detection of the sporozoite-specific Pfslarp and Pfplp1 gene transcripts. These transcripts were chosen based on bioinformatics analysis, and experimentally verified to be overexpressed in the salivary gland sporozoite stage of the parasite compared to other mosquito parasite stages. The proof of principle and the performance of the assay were demonstrated using RNAlater preserved mosquito samples. Tests of analytical sensitivity showed the novel TaqMan assay to be 100% accurate, although its performance in the field needs to be further demonstrated. This method has no requirement for dissection and post-PCR processing and thus is simple and rapid to perform in individual mosquitoes or mosquito pools. It can be used in single or multiplex formats also targeting additional markers expressed in different tissues, such as detoxification enzymes associated with insecticide resistance.",
author = "Mary Kefi and Konstantinos Mavridis and Sim{\~o}es, {Maria L.} and George Dimopoulos and Inga Siden-Kiamos and John Vontas",
note = "Funding Information: Ethics? Statement.? All work was carried out in strict accordance with Greek regulations consisting of the Presidential Decree (160/91) and law (2015/92) which implement the directive 86/609/EEC from the European Union and the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes and the new legislation Presidential Decree 56/2013. The experiments were carried out in a certified animal facility with the license (EL91-BIOexp-02) and the protocol has been approved by the FORTH Committee for Evaluation of Animal Procedures (6740/8/10/2014) and by the Prefecture of Crete (license number # 27290, 15/12/2014). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Care and Use Committee of the Johns Hopkins University (permit number MO15H144). Commercial anonymous human blood was used for parasite cultures and mosquito feeding, and informed consent was therefore not applicable. The Johns Hopkins School of Public Health Ethics Committee has approved this protocol. Funding Information: We are grateful to Edward Thomsen, for helpful discussions and suggestions. We thank Mr Lefteris Spanos for providing mosquitoes and technical support and the Johns Hopkins Malaria Research Institute insectary and parasitology core facilities for assistance with mosquito rearing and preparation of P. falciparum gametocyte cultures. This project was funded by the European Union Horizon 2020 Framework Program (688207 DMC-MALVEC) and was supported by the NIH grants AI061576 and AI081877 and a Johns Hopkins Malaria Research Institute Postdoctoral Fellowship (to Maria L. Sim{\~o}es).",
year = "2018",
month = dec,
day = "1",
doi = "10.1038/s41598-018-19780-6",
language = "English (US)",
volume = "8",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",
}