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Anopheles stephensi mosquitoes are urban malaria vectors in Asia that have recently invaded the Horn of Africa. We detected emergence of An. stephensi mosquitoes in 2 noncontiguous states of eastern Sudan. Results of mitochondrial DNA sequencing suggest the possibility of distinct invasions, potentially from a neighboring country.

Reports of the expansion of the Asia malaria vector Anopheles stephensi mosquito into new geographic areas are increasing, which poses a threat to the elimination of urban malaria. Efficient surveillance of this vector in affected areas and early detection in new geographic areas is key to containing and controlling this species. To overcome the practical difficulties associated with the morphological identification of immature stages and adults of An. stephensi mosquitoes, we developed a species-specific PCR and a real-time PCR targeting a unique segment of the second internal transcribed spacer lacking homology to any other organism. Both PCRs can be used to identify An. stephensi mosquitoes individually or in pooled samples of mixed species, including when present in extremely low proportions (1:500). This study also reports a method for selective amplification and sequencing of partial ribosomal DNA from An. stephensi mosquitoes for their confirmation in pooled samples of mixed species.

Spread of the Anopheles stephensi mosquito, an invasive malaria vector, threatens to put an additional 126 million persons per year in Africa at risk for malaria. To accelerate the early detection and rapid response to this mosquito species, confirming its presence and geographic extent is critical. However, existing molecular species assays require specialized laboratory equipment, interpretation, and sequencing confirmation. We developed and optimized a colorimetric rapid loop-mediated isothermal amplification assay for molecular An. stephensi species identification. The assay requires only a heat source and reagents and can be used with or without DNA extraction, resulting in positive color change in 30-35 minutes. We validated the assay against existing PCR techniques and found 100% specificity and analytical sensitivity down to 0.0003 ng of genomic DNA. The assay can successfully amplify single mosquito legs. Initial testing on samples from Marsabit, Kenya, illustrate its potential as an early vector detection and malaria mitigation tool.

Background: The swift expansion of the invasive malaria vector Anopheles stephensi throughout Africa presents a major challenge to malaria control initiatives. Unlike the native African vectors, An. stephensi thrives in urban settings and has developed resistance to multiple classes of insecticides, including pyrethroids, organophosphates, and carbamates. Methods: Insecticide susceptibility tests were performed on field-collected An. stephensi mosquitoes from Awash Sebac Kilo, Ethiopia, to assess insecticide resistance levels. Illumina RNA-seq analysis was then employed to compare the transcriptomes of field-resistant populations and susceptible laboratory strains (STE2). Results: An. stephensi populations exhibited high levels of resistance to both deltamethrin (mortality, 39.4 ± 6.0%) and permethrin (mortality, 59.3 ± 26.3%) in WHO tube bioassays. RNA-seq analysis revealed that both field-resistant and field-unexposed populations exhibited increased expressions of genes associated with pyrethroid resistance, including esterases, P450s, and GSTs, compared to the susceptible STE2 strain. Notably, esterase E4 and venom carboxylesterase-6 were significantly overexpressed, up to 70-fold, compared to the laboratory strain. Functional enrichment analysis revealed a significant overrepresentation of genes associated with catalytic activity under molecular functions and metabolic process under biological process. Using weighted gene co-expression network analysis (WGCNA), we identified two co-expression modules (green and blue) that included 48 genes strongly linked to pyrethroid insecticide resistance. A co-expression network was subsequently built based on the weight values within these modules. Conclusions: This study highlights the role of esterases in the pyrethroid resistance of an An. stephensi population. The identification of candidate genes associated with insecticide resistance will facilitate the development of rapid diagnostic tools to monitor resistance trends.

In 2012, an unusual outbreak of urban malaria was reported from Djibouti City in the Horn of Africa and increasingly severe outbreaks have been reported annually ever since. Subsequent investigations discovered the presence of an Asian mosquito species; Anopheles stephensi, a species known to thrive in urban environments. Since that first report, An. stephensi has been identified in Ethiopia and Sudan, and this worrying development has prompted the World Health Organization (WHO) to publish a vector alert calling for active mosquito surveillance in the region. Using an up-to-date database of published locational records for An. stephensi across its full range (Asia, Arabian Peninsula, Horn of Africa) and a set of spatial models that identify the environmental conditions that characterize a species’ preferred habitat, we provide evidence-based maps predicting the possible locations across Africa where An. stephensi could establish if allowed to spread unchecked. Unsurprisingly, due to this species’ close association with man-made habitats, our maps predict a high probability of presence within many urban cities across Africa where our estimates suggest that over 126 million people reside. Our results strongly support the WHO’s call for surveillance and targeted vector control and provide a basis for the prioritization of surveillance.

The emergence of the Asian invasive malaria vector, Anopheles stephensi , has been identified in Khartoum, the capital city of Sudan. This is the first report that confirms the geographical expansion of this urban mosquito into Central Sudan. We urgently recommend the launch of a national entomological survey to determine the distribution of this invasive disease vector and to generate essential information about its bionomics and susceptibility to available malaria control measures. Graphical Abstract

The invasive Anopheles stephensi mosquito has rapidly expanded in range in Africa over the past decade. Consistent with World Health Organization guidelines, routine entomologic surveillance of malaria vectors in Accra, Ghana, now includes morphologic and molecular surveillance of An. stephensi mosquitoes. We report detection of An. stephensi mosquitoes in Ghana.

Background Anopheles stephensi  is an efficient vector of both  Plasmodium falciparum  and  Plasmodium vivax  in South Asia and the Middle East. The spread of  An. stephensi  to countries within the Horn of Africa threatens progress in malaria control in this region as well as the rest of sub-Saharan Africa. Methods The available malaria data and the timeline for the detection of An. stephensi was reviewed to analyse the role of  An. stephensi  in malaria transmission in Horn of Africa of the Eastern Mediterranean Region (EMR) in Djibouti, Somalia, Sudan and Yemen. Results Malaria incidence in Horn of Africa of EMR and Yemen, increased from 41.6 in 2015 to 61.5 cases per 1000 in 2020. The four countries from this region, Djibouti, Somalia, Sudan and Yemen had reported the detection of An. stephensi as of 2021. In Djibouti City, following its detection in 2012, the estimated incidence increased from 2.5 cases per 1000 in 2013 to 97.6 cases per 1000 in 2020. However, its contribution to malaria transmission in other major cities and in other countries, is unclear because of other factors, quality of the urban malaria data, human mobility, uncertainty about the actual arrival time of An. stephensi and poor entomological surveillance. Conclusions While An. stephensi may explain a resurgence of malaria in Djibouti, further investigations are needed to understand its interpretation trends in urban malaria across the greater region. More investment for multisectoral approach and integrated surveillance and control should target all vectors particularly malaria and dengue vectors to guide interventions in urban areas.

Anopheles stephensi , an invasive vector is aggressively expanding its geographic range across Africa, posing significant threat to malaria control. Surveillance of its spread is crucial for mitigating its impact on malaria transmission. Here, we report for the first time incursion of An. stephensi into Gayi, a rural area in southern Niger Republic. A combination of morphological identification, end point PCR and sequencing of fragments of COXI and ITS2 genes confirmed An. stephensi . This species was found resting indoor together with other mosquitoes, including An. coluzzii , An. gambiae s.s., and An. arabiensis . Entomological parameters, including resting densities, human blood index and biting rate, Plasmodium falciparum parasite rate, and entomological inoculation rate were described for the above Anopheles species. The finding of An. stephensi sympatric with the above major malaria vectors in Niger highlights the urgent need for intensified surveillance to develop evidence-based approaches to prevent further spread of this pervasive vector.

Background Sub-Saharan Africa has seen substantial reductions in cases and deaths due to malaria over the past two decades. While this reduction is primarily due to an increasing expansion of interventions, urbanisation has played its part as urban areas typically experience substantially less malaria transmission than rural areas. However, this may be partially lost with the invasion and establishment of Anopheles stephensi . A. stephensi , the primary urban malaria vector in Asia, was first detected in Africa in 2012 in Djibouti and was subsequently identified in Ethiopia in 2016, and later in Sudan and Somalia. In Djibouti, malaria cases have increased 30-fold from 2012 to 2019 though the impact in the wider region remains unclear. Methods Here, we have adapted an existing model of mechanistic malaria transmission to estimate the increase in vector density required to explain the trends in malaria cases seen in Djibouti. To account for the observed plasticity in An. stephensi behaviour, and the unknowns of how it will establish in a novel environment, we sample behavioural parameters in order to account for a wide range of uncertainty. This quantification is then applied to Ethiopia, considering temperature-dependent extrinsic incubation periods, pre-existing vector-control interventions and Plasmodium falciparum prevalence in order to assess the potential impact of An. stephensi establishment on P. falciparum transmission. Following this, we estimate the potential impact of scaling up ITN (insecticide-treated nets)/IRS (indoor residual spraying) and implementing piperonyl butoxide (PBO) ITNs and larval source management, as well as their economic costs. Results We estimate that annual P. falciparum malaria cases could increase by 50% (95% CI 14–90) if no additional interventions are implemented. The implementation of sufficient control measures to reduce malaria transmission to pre- stephensi levels will cost hundreds of millions of USD. Conclusions Substantial heterogeneity across the country is predicted and large increases in vector control interventions could be needed to prevent a major public health emergency.