Evidence of Anopheles stephensi involvement in the transmission of Plasmodium vivax in Djibouti City using highly sensitive sporozoite detection assay

BACKGROUND AND OBJECTIVE: 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 country. With the observed increases in human malaria cases and previously reported An. stephensi involvement in outbreaks, high-quality evidence of Plasmodium sporozoite-carrying An. stephensi is essential to confirm and monitor the role of the invasive vector in malaria transmission dynamics in Djibouti. This study aims to detect Plasmodium sporozoites in An. stephensi collected in Djibouti using a multiplex circumsporozoite ELISA bead assay and assess the genetic relatedness of these populations to An. stephensi across the Horn of Africa. METHODS: One hundred and ninety-six wild caught adult An. stephensi mosquitoes from Djibouti City were collected, morphologically identified, and molecularly confirmed using a portion of the cytochrome c oxidase subunit 1 marker. All samples were then tested for infective sporozoites Plasmodium vivax 210, P. vivax 247 and P. falciparum using a multiplex circumsporozoite enzyme-linked immunosorbent assay bead assay. One hundred and fourteen COI sequences that passed further sequence analysis quality control for comparative population genetic analysis were used to characterize the genetic diversity 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 vivax 210 sporozoites were detected in two specimens with a positivity rate of 1.0%. No Pv247 or Pf sporozoites were detected. An analysis of the COI region showed that the Pv210-infected An. stephensi belonged to the dominant invasive haplotypes 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 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, particularly through surveillance and control.