Rao, S.; Samake, J. N.; Rafferty, C.; Mumba, P.; Chibsa, S.; Balkew, M.; Khaireh, B. A.; Guelleh, S. K.; Ibrahim, M. M.; Abdi, A. A.; Zohdy, S.

Purpose: Anopheles stephensi is a malaria mosquito vector that has been raising international concern due to its invasive nature in Africa, including the nation of Djibouti. Since its initial detection in Djibouti in 2012, malaria morbidity and mortality have increased exponentially in the county. While there is an observed association increase in human malaria cases since the arrival of An. stephensi, high-quality evidence of An. stephensi carrying infective sporozoites is essential to determine the role of the invasive vector in malaria dynamics in Djibouti. This study seeks to confirm the link between An. stephensi and malaria transmission in Djibouti and examine genetic relatedness between Djiboutian An. stephensi populations and populations across the Horn of Africa. Such information regarding the An. stephensi populations and the Plasmodium species they transmit is necessary to devise appropriate control strategies and limit malaria transmission within and beyond the country. Methods: One hundred and ninety-six adult An. stephensi mosquitoes from Djibouti were collected, molecularly confirmed, analyzed for a portion of the cytochrome c oxidase subunit 1 (COI), and tested for infective sporozoites using a highly sensitive and specific multiplex circumsporozoite enzyme linked immunosorbent assay (csELISA) bead assay. The COI sequences of one hundred and fourteen samples were further used to characterize the population genetic structure of the sampled An. stephensi and its genetic relatedness to other An. stephensi populations across the Horn of Africa. Results: All 196 samples were morphologically and molecularly confirmed to be An. stephensi. Plasmodium vivax210 sporozoites were detected with a positivity rate of 1.02%. An analysis of the COI region showed that the infected An. stephensi have the most prevalent COI haplotypes of invasive An. stephensi circulating in the Horn of Africa. Conclusions: The findings from this study confirm the involvement of An. stephensi in P. vivax transmission in Djibouti and describe the genetic relatedness of Djiboutian An. stephensi populations to other populations across the Horn of Africa. This highlights the threat of An. stephensi invasion and supports a rapid and comprehensive response to mitigate the harm that An. stephensi populations cause, particularly through surveillance and control of adult populations.