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.

Characterization of Anopheles gambiae D7 salivary proteins as markers of human-mosquito bite contact.

BACKGROUND: Malaria is transmitted when infected Anopheles mosquitoes take a blood meal. During this process, the mosquitoes inject a cocktail of bioactive proteins that elicit antibody responses in humans and could be used as biomarkers of exposure to mosquito bites. This study evaluated the utility of IgG responses to members of the Anopheles gambiae D7 protein family as serological markers of human-vector contact. METHODS: The D7L2, D7r1, D7r2, D7r3, D7r4 and SG6 salivary proteins from An. gambiae were expressed as recombinant antigens in Escherichia coli. Antibody responses to the salivary proteins were compared in Europeans with no prior exposure to malaria and lifelong residents of Junju in Kenya and Kitgum in Uganda where the intensity of malaria transmission is moderate and high, respectively. In addition, to evaluate the feasibility of using anti-D7 IgG responses as a tool to evaluate the impact of vector control interventions, we compared responses between individuals using insecticide-treated bednets to those who did not in Junju, Kenya where bednet data were available. RESULTS: We show that both the long and short forms of the D7 salivary gland antigens elicit a strong antibody response in humans. IgG responses against the D7 antigens reflected the transmission intensities of the three study areas, with the highest to lowest responses observed in Kitgum (northern Uganda), Junju (Kenya) and malaria-naïve Europeans, respectively. Specifically, the long form D7L2 induced an IgG antibody response that increased with age and that was lower in individuals who slept under a bednet, indicating its potential as a serological tool for estimating human-vector contact and monitoring the effectiveness of vector control interventions. CONCLUSIONS: This study reveals that D7L2 salivary antigen has great potential as a biomarker of exposure to mosquito bites and as a tool for assessing the efficacy of vector control strategies such as bednet use.

Physiological and proteomic profiles of Trypanosoma brucei rhodesiense parasite isolated from suramin responsive and non-responsive HAT patients in Busoga, Uganda.

Human African Trypanosomiasis (HAT) is a disease of major economic importance in Sub-Saharan Africa. The HAT is caused by Trypanosoma brucei rhodesiense (Tbr) parasite in eastern and southern Africa, with suramin as drug of choice for treatment of early stage of the disease. Suramin treatment failures has been observed among HAT patients in Tbr foci in Uganda. In this study, we assessed Tbr parasite strains isolated from HAT patients responsive (Tbr EATRO-232) and non-responsive (Tbr EATRO-734) to suramin treatment in Busoga, Uganda for 1) putative role of suramin resistance in the treatment failure 2) correlation of suramin resistance with Tbr pathogenicity and 3) proteomic pathways underpinning the potential suramin resistance phenotype in vivo. We first assessed suramin response in each isolate by infecting male Swiss white mice followed by treatment using a series of suramin doses. We then assessed relative pathogenicity of the two Tbr isolates by assessing changes pathogenicity indices (prepatent period, survival and mortality). We finally isolated proteins from mice infected by the isolates, and assessed their proteomic profiles using mass spectrometry. We established putative resistance to 2.5 mg/kg suramin in the parasite Tbr EATRO-734. We established that Tbr EATRO-734 proliferated slower and has significantly enriched pathways associated with detoxification and metabolism of energy and drugs relative to Tbr EATRO-232. The Tbr EATRO-734 also has more abundantly expressed mitochondrion proteins and enzymes than Tbr EATRO-232. The suramin treatment failure may be linked to the relatively higher resistance to suramin in Tbr EATRO-734 than Tbr EATRO-232, among other host and parasite specific factors. However, the Tbr EATRO-734 appears to be less pathogenic than Tbr EATRO-232, as evidenced by its lower rate of parasitaemia. The Tbr EATRO-734 putatively surmount suramin challenges through induction of energy metabolism pathways. These cellular and molecular processes may be involved in suramin resistance in Tbr.

Bionomics and ecology of Anopheles merus along the East and Southern Africa coast.

Malaria transmission persists despite the scale-up of interventions such as long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS). Understanding the entomological drivers of transmission is key for the design of effective and sustainable tools to address the challenge. Recent research findings indicate a shift in vector populations from the notorious Anopheles gambiae (s.s.) as a dominant vector to other species as one of the factors contributing to the persistence of malaria transmission. However, there are gaps in the literature regarding the minor vector species which are increasingly taking a lead role in malaria transmission. Currently, minor malaria vectors have behavioural plasticity, which allows their evasion of vector control tools currently in use. To address this, we have reviewed the role of Anopheles merus, a saltwater mosquito species that is becoming an important vector of malaria transmission along the East and Southern African coast. We performed a literature review from PubMed and Google Scholar and reviewed over 50 publications relating to An. merus's bionomics, taxonomy, spatial-temporal distribution and role in malaria transmission. We found that An. merus is an important vector of malaria and that it contributes to residual malaria transmission because of its exophilic tendencies, insecticide resistance and densities that peak during the dry seasons as the freshwater mosquitoes decline. Spatial and temporal studies have also shown that this species has increased its geographical range, densities and vectorial capacity over time. In this review, we highlight the resting behaviour and breeding habitats of this mosquito, which could be targeted for surveillance studies and control interventions.

Expansions of chemosensory gene orthologs among selected tsetse fly species and their expressions in Glossina morsitans morsitans tsetse fly.

Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7) expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d, Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b, GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes in the head were associated with vision, muscle activity and neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments (>90% of chemosensory transcripts) included cilium-associated mechanoreceptors, chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to stress. The expanded and tsetse fly species specific orthologs includes those associated with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid, butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2-oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine, geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking, cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and kairomones for respective downstream functional genomic and field evaluations that can effectively expand toolbox of species-specific tsetse fly attractants, repellents and other tsetse fly behavioral modulators.

Peripheral blood mononuclear cell transcriptomes reveal an over-representation of down-regulated genes associated with immunity in HIV-exposed uninfected infants.

HIV-exposed uninfected (HEU) infants are disproportionately at a higher risk of morbidity and mortality, as compared to HIV-unexposed uninfected (HUU) infants. Here, we used transcriptional profiling of peripheral blood mononuclear cells to determine immunological signatures of in utero HIV exposure. We identified 262 differentially expressed genes (DEGs) in HEU compared to HUU infants. Weighted gene co-expression network analysis (WGCNA) identified six modules that had significant associations with clinical traits. Functional enrichment analysis on both DEGs and the six significantly associated modules revealed an enrichment of G-protein coupled receptors and the immune system, specifically affecting neutrophil function and antibacterial responses. Additionally, malaria pathogenicity genes (thrombospondin 1-(THBS 1), interleukin 6 (IL6), and arginine decarboxylase 2 (ADC2)) were down-regulated. Of interest, the down-regulated immunity genes were positively correlated to the expression of epigenetic factors of the histone family and high-mobility group protein B2 (HMGB2), suggesting their role in the dysregulation of the HEU transcriptional landscape. Overall, we show that genes primarily associated with neutrophil mediated immunity were repressed in the HEU infants. Our results suggest that this could be a contributing factor to the increased susceptibility to bacterial infections associated with higher morbidity and mortality commonly reported in HEU infants.

Cadmium tolerance pathway in Anopheles gambiae senso stricto.

Cadmium is one of the widely used heavy metals (HM) in commercial and industrial products and contributes to environmental contamination in an urban setting. In our previous studies, we established that An. gambiae sensu stricto, a vector of malaria, had adapted to HM pollutants in nature despite their proclivity for unpolluted aquatic habitats. We further demonstrated that heavy metal tolerance adaptation process impacts a biological cost to the fitness of the mosquito and potentially involves the induction of specific HM-responsive transcripts and proteins. Here we interrogated differential proteomic profiles of the cadmium tolerant vs. naïve strains of An. gambiae to shed light on proteomic processes that underpinned biological cost to fitness. We identified a total of 1067 larval proteins and observed significant down-regulation of proteins involved in larval immune responses, energy metabolism, antioxidant enzymes, protein synthesis, and proton transport. Our results suggest that mosquitoes can adjust their biological program through proteome changes to counter HM pollution. Since our study was done in controlled laboratory settings, we acknowledge this may not wholly represent the conditions HM polluted environments. Nevertheless, mosquitoes deploying this strategy have the potential of creating an urban enclave for breeding and thrive and become agents of sporadic malaria epidemics.

Mosquito microevolution drives Plasmodium falciparum dynamics.

Malaria, a major cause of child mortality in Africa, is engendered by Plasmodium parasites that are transmitted by anopheline mosquitoes. Fitness of Plasmodium parasites is closely linked to the ecology and evolution of its anopheline vector. However, whether the genetic structure of vector populations impacts malaria transmission remains unknown. Here, we describe a partitioning of the African malaria vectors into generalists and specialists that evolve along ecological boundaries. We next identify the contribution of mosquito species to Plasmodium abundance using Granger causality tests for time-series data collected over two rainy seasons in Mali. We find that mosquito microevolution, defined by changes in the genetic structure of a population over short ecological timescales, drives Plasmodium dynamics in nature, whereas vector abundance, infection prevalence, temperature and rain have low predictive values. Our study demonstrates the power of time-series approaches in vector biology and highlights the importance of focusing local vector control strategies on mosquito species that drive malaria dynamics.

Growth-disrupting Murraya koenigii leaf extracts on Anopheles gambiae larvae and identification of associated candidate bioactive constituents.

Plant-based constituents have been proposed as eco-friendly alternatives to synthetic insecticides for control of mosquito vectors of malaria. In this study, we first screened the effects of methanolic leaf extracts of curry tree (Murraya koenigii) growing in tropical (Mombasa, Malindi) and semi-arid (Kibwezi, and Makindu) ecological zones of Kenya on third instar An. gambiae s.s. larvae. Extracts of the plant from the semi-arid region, and particularly from Kibwezi, led to high mortality of the larvae. Bioassay-guided fractionation of the methanolic extract of the leaves of the plants from Kibwezi was then undertaken and the most active fraction (20 fold more potent than the crude extract) was then analyzed by Liquid chromatography quadruple time of flight coupled with mass spectrometry (LC-QtoF-MS) and a number of constituents were identified, including a major alkaloid constituent, Neplanocin A (5). Exposure of the third instar larvae to a sub-lethal dose (4.43 ppm) of this fraction over 7-day periods induced gross morphogenetic abnormalities in the larvae, with reduced locomotion, and delayed pupation. Moreover, the few adults that emerged from some pupae failed to fly from the water surface, unlike in the untreated control group. These results demonstrate subtle growth-disrupting effects of the phytochemical blend from M. koenigii leaves on aquatic stages An. gambiae mosquito. The study lays down some useful groundwork for the downstream development of phytochemical blends that can be evaluated for integration into eco-friendly control of An. gambiae vector population targeting the often overlooked but important immature stages of the malaria vector.

Adaptation of Plasmodium falciparum to its transmission environment.

Success in eliminating malaria will depend on whether parasite evolution outpaces control efforts. Here, we show that Plasmodium falciparum parasites (the deadliest of the species causing human malaria) found in low-transmission-intensity areas have evolved to invest more in transmission to new hosts (reproduction) and less in within-host replication (growth) than parasites found in high-transmission areas. At the cellular level, this adaptation manifests as increased production of reproductive forms (gametocytes) early in the infection at the expense of processes associated with multiplication inside red blood cells, especially membrane transport and protein trafficking. At the molecular level, this manifests as changes in the expression levels of genes encoding epigenetic and translational machinery. Specifically, expression levels of the gene encoding AP2-G-the transcription factor that initiates reproduction-increase as transmission intensity decreases. This is accompanied by downregulation and upregulation of genes encoding HDAC1 and HDA1-two histone deacetylases that epigenetically regulate the parasite's replicative and reproductive life-stage programmes, respectively. Parasites in reproductive mode show increased reliance on the prokaryotic translation machinery found inside the plastid-derived organelles. Thus, our dissection of the parasite's adaptive regulatory architecture has identified new potential molecular targets for malaria control.

Cerebrospinal fluid markers to distinguish bacterial meningitis from cerebral malaria in children.

Background. Few hospitals in high malaria endemic countries in Africa have the diagnostic capacity for clinically distinguishing acute bacterial meningitis (ABM) from cerebral malaria (CM). As a result, empirical use of antibiotics is necessary. A biochemical marker of ABM would facilitate precise clinical diagnosis and management of these infections and enable rational use of antibiotics. Methods. We used label-free protein quantification by mass spectrometry to identify cerebrospinal fluid (CSF) markers that distinguish ABM (n=37) from CM (n=22) in Kenyan children. Fold change (FC) and false discovery rates (FDR) were used to identify differentially expressed proteins. Subsequently, potential biomarkers were assessed for their ability to discriminate between ABM and CM using receiver operating characteristic (ROC) curves. Results. The host CSF proteome response to ABM ( Haemophilusinfluenza and Streptococcuspneumoniae) is significantly different to CM. Fifty two proteins were differentially expressed (FDR<0.01, Log FC≥2), of which 83% (43/52) were upregulated in ABM compared to CM. Myeloperoxidase and lactotransferrin were present in 37 (100%) and 36 (97%) of ABM cases, respectively, but absent in CM (n=22). Area under the ROC curve (AUC), sensitivity, and specificity were assessed for myeloperoxidase (1, 1, and 1; 95% CI, 1-1) and lactotransferrin (0.98, 0.97, and 1; 95% CI, 0.96-1). Conclusion. Myeloperoxidase and lactotransferrin have a high potential to distinguish ABM from CM and thereby improve clinical management. Their validation requires a larger cohort of samples that includes other bacterial aetiologies of ABM.

Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome.

BACKGROUND: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. METHODS: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). RESULTS: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. CONCLUSIONS: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.

Variation in the effectiveness of insecticide treated nets against malaria and outdoor biting by vectors in Kilifi, Kenya.

Background: Insecticide treated nets (ITNs) protect humans against bites from the Anopheles mosquito vectors that transmit malaria, thereby reducing malaria morbidity and mortality. It has been noted that ITN use leads to a switch from indoor to outdoor feeding among these vectors. It might be expected that outdoor feeding would undermine the effectiveness of ITNs that target indoors vectors, but data are limited. Methods: We linked homestead level geospatial data to clinical surveillance data at a primary healthcare facility in Kilifi County in order to map geographical heterogeneity in ITN effectiveness and observed vector feeding behaviour using landing catches and CDC light traps in six selected areas of varying ITN effectiveness. We quantified the interaction between mosquitoes and humans to evaluate whether outdoor vector biting is a potential explanation for the variation in ITN effectiveness. Results: We observed 37% and 46% visits associated with positive malaria slides among ITN users and non-ITN-users, respectively; ITN use was associated with 32% protection from malaria (crude OR = 0.68, 95% CI: 0.64, 0.73). We obtained significant modification of ITN effectiveness by geographical area (p=0.016), and identified significant hotspots using the spatial scan statistic. Majority of mosquitoes were caught outdoor (60%) and were of the An. funestus group (75%). The overall propensity to feed at times when most people are indoor was high; the vast majority of the Anopheles mosquitoes were caught at times when most people are indoor. Estimates for the proportion of human-mosquito contact between the first and last hour when most humans were indoor was consistently high, ranging from 0.83 to 1.00. Conclusion: Our data do not provide evidence of an epidemiological association between microgeographical variations in ITN effectiveness and variations in the microgeographical distribution of outdoor biting.

CD26/dipeptidyl peptidase IV (CD26/DPPIV) is highly expressed in peripheral blood of HIV-1 exposed uninfected female sex workers.

BACKGROUND: Design of effective vaccines against the human immunodeficiency virus (HIV-1) continues to present formidable challenges. However, individuals who are exposed HIV-1 but do not get infected may reveal correlates of protection that may inform on effective vaccine design. A preliminary gene expression analysis of HIV resistant female sex workers (HIV-R) suggested a high expression CD26/DPPIV gene. Previous studies have indicated an anti-HIV effect of high CD26/DPPIV expressing cells in vitro. Similarly, high CD26/DPPIV protein levels in vivo have been shown to be a risk factor for type 2 diabetes. We carried out a study to confirm if the high CD26/DPPIV gene expression among the HIV-R were concordant with high blood protein levels and its correlation with clinical type 2 diabetes and other perturbations in the insulin signaling pathway. RESULTS: A quantitative CD26/DPPIV plasma analysis from 100 HIV-R, 100 HIV infected (HIV +) and 100 HIV negative controls (HIV Neg) showed a significantly elevated CD26/DPPIV concentration among the HIV-R group (mean 1315 ng/ml) than the HIV Neg (910 ng/ml) and HIV + (870 ng/ml, p < 0.001). Similarly a FACs analysis of cell associated DPPIV (CD26) revealed a higher CD26/DPPIV expression on CD4+ T-cells derived from HIV-R than from the HIV+ (90.30% vs 80.90 p = 0.002) and HIV Neg controls (90.30% vs 82.30 p < 0.001) respectively. A further comparison of the mean fluorescent intensity (MFI) of CD26/DPPIV expression showed a higher DPP4 MFI on HIV-R CD4+ T cells (median 118 vs 91 for HIV-Neg, p = 0.0003). An evaluation for hyperglycemia, did not confirm Type 2 diabetes but an impaired fasting glucose condition (5.775 mmol/L). A follow-up quantitative PCR analysis of the insulin signaling pathway genes showed a down expression of NFκB, a central mediator of the immune response and activator of HIV-1 transcription. CONCLUSION: HIV resistant sex workers have a high expression of CD26/DPPIV in tandem with lowered immune activation markers. This may suggest a novel role for CD26/DPPIV in protection against HIV infection in vivo.

The major yolk protein vitellogenin interferes with the anti-plasmodium response in the malaria mosquito Anopheles gambiae.

When taking a blood meal on a person infected with malaria, female Anopheles gambiae mosquitoes, the major vector of human malaria, acquire nutrients that will activate egg development (oogenesis) in their ovaries. Simultaneously, they infect themselves with the malaria parasite. On traversing the mosquito midgut epithelium, invading Plasmodium ookinetes are met with a potent innate immune response predominantly controlled by mosquito blood cells. Whether the concomitant processes of mosquito reproduction and immunity affect each other remains controversial. Here, we show that proteins that deliver nutrients to maturing mosquito oocytes interfere with the antiparasitic response. Lipophorin (Lp) and vitellogenin (Vg), two nutrient transport proteins, reduce the parasite-killing efficiency of the antiparasitic factor TEP1. In the absence of either nutrient transport protein, TEP1 binding to the ookinete surface becomes more efficient. We also show that Lp is required for the normal expression of Vg, and for later Plasmodium development at the oocyst stage. Furthermore, our results uncover an inhibitory role of the Cactus/REL1/REL2 signaling cassette in the expression of Vg, but not of Lp. We reveal molecular links that connect reproduction and immunity at several levels and provide a molecular basis for a long-suspected trade-off between these two processes.