Until 2020, Djiboutian health authorities relied on histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) to establish the diagnosis of Plasmodium falciparum. The rapid spread of P. falciparum histidine-rich protein-2 and -3 (pfhrp2/3) gene-deleted parasite strains in Djibouti has led the authorities to switch from HRP2-based RDTs to lactate dehydrogenase (LDH)-based RDTs targeting the plasmodial lactate dehydrogenase (pLDH) specific for P. falciparum and P. vivax (RapiGEN BIOCREDIT Malaria Ag Pf/Pv pLDH/pLDH) in 2021. This study was conducted with the primary objective of evaluating the diagnostic performance of this alternative RDT. Operational constraints related, in particular, to the implementation of this RDT during the COVID-19 pandemic were also considered. The performance of BIOCREDIT Malaria Ag Pf/Pv (pLDH/pLDH) RDT was also compared to our previously published data on the performance of two HRP2-based RDTs deployed in Djibouti in 2018–2020. The diagnosis of 350 febrile patients with suspected malaria in Djibouti city was established using two batches of RapiGEN BIOCREDIT Malaria Ag Pf/Pv (pLDH/pLDH) RDT over a two-year period (2022 and 2023) and confirmed by real-time quantitative polymerase chain reaction. The sensitivity and specificity for the detection of P. falciparum were 88.2% and 100%, respectively. For P. vivax, the sensitivity was 86.7% and the specificity was 100%. Re-training and closer supervision of the technicians between 2022 and 2023 have led to an increased sensitivity to detect P. falciparum (69.8% in 2022 versus 88.2% in 2023; p < 0.01). The receiver operating characteristic curve analysis highlighted a better performance in the diagnosis of P. falciparum with pLDH-based RDTs compared with previous HRP2-based RDTs. In Djibouti, where pfhrp2-deleted strains are rapidly gaining ground, LDH-based RDTs seem to be more suitable for diagnosing P. falciparum than HRP2-based RDTs. Awareness-raising and training for technical staff have also been beneficial.

Historically, native populations in the Republic of Djibouti have experienced only low and unstable malaria transmission and intermittent epidemics. In recent years, efforts at malaria control have been aggressively pursued. This study was performed to inform revised malaria prevention recommendations for military service members and international travelers to the country. Laboratory-confirmed cases of malaria documented at large medical facilities and within military and civilian health care systems in the Republic of Djibouti from 1998 to 2009 were reviewed. In recent years, fewer than 5% of febrile cases among the three largest passive surveillance systems were laboratory-confirmed as malaria, and incidence of confirmed malaria was well below 1/1,000 persons/year. As efforts in the Republic of Djibouti progress toward elimination, and in conjunction with continued efforts at surveillance, emphasizing mosquito-avoidance measures and standby emergency treatment will become reasonable recommendations for malaria prevention.

The invasive urban malaria vector Anopheles stephensi threatens 126 million city dwellers in Africa. Controlling An. stephensi requires greater understanding of its origin, invasion dynamics, and insecticide resistance mechanisms. Analysis of 645 whole genomes sampled across Africa, the Middle East, and Asia supports an invasion scenario in which an initial South Asian introduction established a bridgehead population in Djibouti, which seeded distinct invasion fronts in Sudan, Ethiopia/Kenya, and Yemen. These incursions show contrasting rates and routes of spread shaped by landscape topology. Insecticide resistance is predominantly mediated by metabolic detoxification genes, with resistance haplotypes and copy-number amplifications introduced from South Asia. These findings, alongside a companion genomic resource, enable genomic surveillance of An. stephensi spread and resistance to aid control strategies.

The invasion of Africa by the Asian urban malaria vector, Anopheles stephensi, endangers 126 million people across a rapidly urbanising continent where malaria is primarily a rural disease. Control of An. stephensi requires greater understanding of its origin, invasion dynamics, and mechanisms of widespread resistance to vector control insecticides. We present a genomic surveillance study of 551 An. stephensi sampled across the invasive and native ranges in Africa and Asia. Our findings support a hypothesis that an initial invasion from Asia to Djibouti seeded separate incursions to Sudan, Ethiopia, and Yemen before spreading inland, aided by favourable temperature, vegetation cover, and human transit conditions. Insecticide resistance in invasive An. stephensi is conferred by detoxification genes introduced from Asia. These findings, and a companion genomic data catalogue, will form the foundation of an evidence base for surveillance and management strategies for An. stephensi .

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 vivax 210 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. Graphical

Djibouti is confronted with malaria resurgence, with malaria having been occurring in epidemic proportions since a decade ago. The current epidemiology of drug-resistant Plasmodium falciparum is not well known. Molecular markers were analyzed by targeted sequencing in 79 P. falciparum clinical isolates collected in Djibouti city in 2023 using the Miseq Illumina platform newly installed in the country. The objective of the study was to analyze the key codons in these molecular markers associated with antimalarial drug resistance. The prevalence of the mutant Pfcrt CVIET haplotype (92%) associated with chloroquine resistance and mutant Pfdhps-Pfdhfr haplotypes (7.4% SGEA and 53.5% IRN, respectively) associated with sulfadoxine-pyrimethamine resistance was high. By contrast, Pfmdr1 haplotypes associated with amodiaquine (YYY) or lumefantrine (NFD) resistance were not observed in any of the isolates. Although the “Asian-type” PfK13 mutations associated with artemisinin resistance were not observed, the “African-type” PfK13 substitution, R622I, was found in a single isolate (1.4%) for the first time in Djibouti. Our genotyping data suggest that most Djiboutian P. falciparum isolates are resistant to chloroquine and sulfadoxine-pyrimethamine but are sensitive to amodiaquine, lumefantrine, and artemisinin. Nonetheless, the presence of an isolate with the R622I PfK13 substitution is a warning signal that calls for a regular surveillance of molecular markers of antimalarial drug resistance.