Loop-Mediated Isothermal Amplification Assay to Detect Invasive Malaria Vector Anopheles stephensi Mosquitoes.

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 nanograms 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.

Detection of Anopheles stephensi Mosquitoes by Molecular Surveillance, Kenya.

The Anopheles stephensi mosquito is an invasive malaria vector recently reported in Djibouti, Ethiopia, Sudan, Somalia, Nigeria, and Ghana. The World Health Organization has called on countries in Africa to increase surveillance efforts to detect and report this vector and institute appropriate and effective control mechanisms. In Kenya, the Division of National Malaria Program conducted entomological surveillance in counties at risk for An. stephensi mosquito invasion. In addition, the Kenya Medical Research Institute conducted molecular surveillance of all sampled Anopheles mosquitoes from other studies to identify An. stephensi mosquitoes. We report the detection and confirmation of An. stephensi mosquitoes in Marsabit and Turkana Counties by using endpoint PCR and morphological and sequence identification. We demonstrate the urgent need for intensified entomological surveillance in all areas at risk for An. stephensi mosquito invasion, to clarify its occurrence and distribution and develop tailored approaches to prevent further spread.

Serotype and genetic diversity of human rhinovirus strains that circulated in Kenya in 2008.

BACKGROUND: Human rhinoviruses (HRVs) are a well-established cause of the common cold and recent studies indicated that they may be associated with severe acute respiratory illnesses (SARIs) like pneumonia, asthma, and bronchiolitis. Despite global studies on the genetic diversity of the virus, the serotype diversity of these viruses across diverse geographic regions in Kenya has not been characterized. OBJECTIVES: This study sought to characterize the serotype diversity of HRV strains that circulated in Kenya in 2008. METHODS: A total of 517 archived nasopharyngeal samples collected in a previous respiratory virus surveillance program across Kenya in 2008 were selected. Participants enrolled were outpatients who presented with influenza-like (ILI) symptoms. Real-time RT-PCR was employed for preliminary HRV detection. HRV-positive samples were amplified using RT-PCR and thereafter the nucleotide sequences of the amplicons were determined followed by phylogenetic analysis. RESULTS: Twenty-five percent of the samples tested positive for HRV. Phylogenetic analysis revealed that the Kenyan HRVs clustered into three main species comprising HRV-A (54%), HRV-B (12%), and HRV-C (35%). Overall, 20 different serotypes were identified. Intrastrain sequence homology among the Kenyan strains ranged from 58% to 100% at the nucleotide level and 55% to 100% at the amino acid level. CONCLUSION: These results show that a wide range of HRV serotypes with different levels of nucleotide variation were present in Kenya. Furthermore, our data show that HRVs contributed substantially to influenza-like illness in Kenya in 2008.