Stalin Vilcarromero, Crystyan Siles, Eric S. Halsey, Leslye Angulo, Julia Schwarz, Cesar Ramal-Asayag, Louis Lambrechts, Kanya C. Long, Thomas W. Scott, Karin S. Escobedo-Vargas, Hugo L. Jaba, Robert D. Hontz, Juan Sulca, Valerie A. Paz-Soldan, Amy C. Morrison, Guadalupe Flores, Fanny Castro-Llanos, Claudine Kocher, Isabel Bazan, Helvio Astete, Horstick, Olaf, University of California [Davis] (UC Davis), University of California, U.S. Naval Medical Research Unit No. 6 (NAMRU-6), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Universidad Nacional de la Amazonía Peruana [Loreto, Perou] (UNAP), Tulane University, Interactions Virus-Insectes - Insect-Virus Interactions (IVI), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], We thank the Parker Huang Undergraduate Travel Fellowship from Yale University for funding JS while working on this project. This research was funded by two grants from the U.S. National Institutes of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID): principally, grant award number R03 AI107446-01 (to A.C.M. and L.L.), and, secondarily, grant award numbers R01 AI069341 and P01 AI098670 (to T.W.S.)., We thank the residents of Iquitos for their participation in this study. We greatly appreciate support of the Loreto Regional Health Department, including Drs. Hugo Rodriguez-Ferruci, Christian Carey, Carlos Alvarez, Hernan Silva, and Lic. Wilma Casanova Rojas, who all facilitated our work in Iquitos. Christopher Mores, Cecilia Gonzales, Kyle Peterson, Adam Armstrong, and Guillermo Pimentel, provided institutional support through NAMRU-6, and Maria Silva and Carolina Guevara supervised laboratory testing. A special thanks to Gloria Talledo for her ongoing support with the preparation of IRB protocols and reports for this project. We also thank Alan Lozano, Rebecca Carrion, and Geovana Hora for their help with development of the consent video. We also appreciate the careful commentary and advice provided by the NAMRU-6 IRB and Research Administration Program for the duration of this study, and especially that provided by Roxana Lescano, Zoe Moran, and Toane Zuleta. Regina Fernandez supervised field teams, which included Llerme Armas, Karina Chuquipiondo, Leny Curico, Rocio Del Rio, Junnelhy Flores, Juan Flores, Luz Angelica Galvez, Rina Gonzales, Maria Edith Juarez, Xiomara Mafaldo, Nora Marin, Nadia Montes, Johnni Mozombite, Sandra Munoz, Lucy Navarro, Geraldine Ocmin, Zenith Pezo, Iris Reategui, Sadith Jovita Ricopa, Liliana Rios, Rubiela Rubio, Ysabel Ruis, Rosana Sotero, Rosa Tamani, Zenith Tamani, and Sarita Del Pilar Tuesta, to identify acute dengue cases. We thank Gabriela Vasquez de la Torre for her administrative support for the project., University of California (UC), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Background Transmission of dengue virus (DENV) from humans to mosquitoes represents a critical component of dengue epidemiology. Examinations of this process have generally been hampered by a lack of methods that adequately represent natural acquisition of DENV by mosquitoes from humans. In this study, we assessed artificial and natural blood feeding methods based on rates of DENV infection and dissemination within mosquitoes for use in a field-based epidemiological cohort study in Iquitos, Peru. Methodology/Principal findings Our study was implemented, stepwise, between 2011 and 2015. Participants who were 5 years and older with 5 or fewer days of fever were enrolled from ongoing clinic- and neighborhood-based studies on dengue in Iquitos. Wild type, laboratory-reared Aedes aegypti were fed directly on febrile individuals or on blood collected from participants that was either untreated or treated with EDTA. Mosquitoes were tested after approximately 14 days of extrinsic incubation for DENV infection and dissemination. A total of 58 participants, with viremias ranging from 1.3 × 102 to 2.9 × 106 focus-forming units per mL of serum, participated in one or more feeding methods. DENV infection and dissemination rates were not significantly different following direct and indirect-EDTA feeding; however, they were significantly lower for mosquitoes that fed indirectly on blood with no additive. Relative to direct feeding, infection rates showed greater variation following indirect-EDTA than indirect-no additive feeding. Dissemination rates were similar across all feeding methods. No differences were detected in DENV infection or dissemination rates in mosquitoes fed directly on participants with different dengue illness severity. Conclusions/Significance Our study demonstrates the feasibility of using direct and indirect feeding methods for field-based studies on vector competence. Direct mosquito feeding is preferable in terms of logistical ease, biosecurity, and reliability., Author summary In the context of ongoing clinic- and field-based epidemiological studies on dengue virus (DENV), we compared methods of feeding mosquitoes on blood from naturally infected humans. Participants chose to participate in direct (uninfected mosquitoes applied directly to skin) and/or indirect (uninfected mosquitoes fed on a membrane feeder in the laboratory with blood drawn from a participant) methods. Overall, rates of DENV infection and dissemination were lower in mosquitoes fed indirectly on blood with no additive than in mosquitoes fed directly on a participant. Rates of DENV infection and dissemination were similar between mosquitoes fed directly and indirectly when anticoagulant (EDTA) was added. The indirect-EDTA method resulted in more variable infection rates than the direct method. Our results demonstrate the feasibility of using either direct or indirect feeding methods to study DENV-human infectiousness to mosquito vectors. In our experience, however, direct feeding is preferable to indirect feeding in terms of logistical ease, biosecurity, and reliability.