Ethiopian Plasmodium vivax hypnozoites formation dynamics and their susceptibility to reference antimalarial drugs.

One of the key obstacles to malaria elimination is largely attributed to Plasmodium vivax's ability to form resilient hypnozoites in the host liver that cause relapsing infections. As a result, interruption of P. vivax transmission is difficult. P. vivax transmission occurs in Duffy-positive individuals and have been mainly thought to be absent in Africa. However, increasing studies using molecular tools detected P. vivax among Duffy-negative individuals in various African countries. Studies on the African P. vivax has been severely limited because most of malaria control program focus mainly on falciparum malaria. In addition, there is a scarcity of laboratory infrastructures to overcome the biological obstacles posed by P. vivax. Herein, we established field transmission of Ethiopian P. vivax for routine sporozoite supply followed by liver stage infection in Mali. Furthermore, we evaluated local P. vivax hypnozoites and schizonts susceptibilities to reference antimalarial drugs. The study enabled the assessment of local African P. vivax hypnozoite production dynamics. Our data displayed the ability of the African P. vivax to produce hypnozoite forms ex-vivo at different rates per field isolate. We report that while tafenoquine (1µM) potently inhibited both hypnozoites and schizont forms; atovaquone (0.25µM) and the phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691 (0.5µM) showed no activity against hypnozoites forms. Unlike hypnozoites forms, P. vivax schizont stages were fully susceptible to both atovaquone (0.25µM) and the (PI4K)-specific inhibitor KDU691 (0.5µM). Together, the data revealed the importance of the local platform for further biological investigation and implementation of drug discovery program on the African P. vivax clinical isolates.

A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Identification of mixed and successive blood meals of mosquitoes using MALDI-TOF MS protein profiling.

BACKGROUND: The accurate and rapid identification of mosquito blood meals is critical to study the interactions between vectors and vertebrate hosts and, subsequently, to develop vector control strategies. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has been shown to be a reliable and effective tool for identifying single blood meals from mosquitoes. METHODS: In this study, we developed MALDI-TOF MS profiling protocols to identify Anopheles gambiae Giles, Anopheles coluzzii and Aedes albopictus mosquitoes' mixed blood meals and the last of successive blood meals. The mosquitoes were either successively artificially fed with distinct host bloods or engorged with mixed bloods from distinct vertebrate hosts, such as humans, sheep and dogs. RESULTS: Blind test analyses revealed a correct identification of mixed blood meals from mosquitoes using MALDI-TOF MS profiling. The 353 MS spectra from mixed blood meals were identified using log score values >1.8. All MS spectra (n = 244) obtained from mosquitoes' successive blood meals were reproducible and specific to the last blood meal, suggesting that the previous blood meals do not have an impact on the identification of the last one. CONCLUSION: MALDI-TOF MS profiling approach appears to be an effective and robust technique to identify the last and mixed blood meals during medical entomological surveys.

Mosquitoes (Diptera: Culicidae) and mosquito-borne diseases in Mali, West Africa.

Mosquito-borne diseases cause major human diseases in almost every part of the world. In West Africa, and notably in Mali, vector control measures help reduce the impact of mosquito-borne diseases, although malaria remains a threat to both morbidity and mortality. The most recent overview article on mosquitoes in Mali was published in 1961, with a total of 88 species. Our present review focuses on mosquitoes of medical importance among which the Anopheles vectors of Plasmodium and filaria, as well as the Culex and Aedes vectors of arboviruses. It aims to provide a concise update of the literature on Culicidae, covering the ecological areas in which the species are found but also the transmitted pathogens and recent innovative tools for vector surveys. This review highlights the recent introduction of invasive mosquito species, including Aedes albopictus and Culex neavei. The comprehensive list of mosquito species currently recorded includes 106 species (28 species of the Anophelinae and 78 species of the Culicinae). There are probable gaps in our knowledge concerning mosquitoes of the subfamily Culicinae and northern half of Mali because most studies have been carried out on the genus Anopheles and have taken place in the southern part of the country. It is hoped that this review may be useful to decision makers responsible for vector control strategies and to researchers for future surveys on mosquitoes, particularly the vectors of emerging arboviruses.

Blood meal identification in the cryptic species Anopheles gambiae and Anopheles coluzzii using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has recently emerged in entomology as a technique to identify arthropods and their blood meal source. In this study, female Anopheles gambiae were fed on five host blood sources: ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) and Hamadryas baboon (Papio hamadryas), while Anopheles coluzzii were fed on three hosts: dromedary (Camelus dromedarius), Barbary sheep (Ammotragus lervia) and pig (Sus scrofa). We obtained the MS spectra from 240 engorged mosquito abdomens and selected high quality ones from 72 mosquito abdomens to upgrade our home-made database. We excluded from the analysis any spectra of low quality (n = 80), and the remaining 88 specimens were subjected to a blind test analysis against the home-made database. We obtained 100% correct identification of the blood meal source for the specimens collected, 1, 12 and 24 h post-feeding, whereas for the specimens collected 36 h post-feeding, the correct identification rate decreased dramatically. We confirm here that MALDI-TOF MS can be used to identify the blood meal origin of freshly engorged mosquitoes, which opens new perspectives for further studies, including the impact of the mosquito species on blood meal identification.

TITLE: Identification du repas sanguin des espèces cryptiques Anopheles gambiae et Anopheles coluzzii par l'utilisation de MALDI-TOF MS. ABSTRACT: L'identification par spectrométrie de masse à temps de vol par désorption/ionisation assistée par matrice (MALDI-TOF MS) a récemment émergé en entomologie pour l'identification des arthropodes et de leur source de sang. Des femelles d'Anopheles gambiae ont été nourries de sang de cinq hôtes, ocelot (Leopardus pardalis), binturong (Arctictis binturong), springbok (Antidorcas marsupialis), jaguar (Panthera onca) et babouin Hamadryas (Papio hamadryas), et des femelles d'Anopheles coluzzii ont été nourries sur trois hôtes, dromadaire (Camelus dromedarius), mouflon à manchettes (Ammotragus lervia) et porc (Sus scrofa). Nous avons obtenu les spectres MS à partir de 240 abdomens de moustiques engorgés et avons sélectionné ceux de 72 abdomens de moustiques de haute qualité pour améliorer notre base de données maison. Nous avons exclu de l'analyse les spectres de faible qualité (n = 80) et les 88 échantillons restants ont été soumis à une analyse de test en aveugle contre la base de données maison. Nous avons obtenu 100 % d'identification correcte de la source de sang pour les échantillons collectés, 1, 12 et 24 heures après l'alimentation, mais le taux d'identification correct a diminué de façon spectaculaire pour les échantillons collectés 36 heures après l'alimentation. Nous confirmons ici que la MALDI-TOF MS peut être utilisée pour identifier l'origine des repas sanguins des moustiques fraîchement engorgés et ouvre de nouvelles perspectives pour d'autres études, y compris l'impact des espèces de moustiques sur l'identification des repas sanguins.

Using MALDI-TOF MS to identify mosquitoes collected in Mali and their blood meals.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently described as an innovative and effective tool for identifying arthropods and mosquito blood meal sources. To test this approach in the context of an entomological survey in the field, mosquitoes were collected from five ecologically distinct areas of Mali. We successfully analysed the blood meals from 651 mosquito abdomens crushed on Whatman filter paper (WFPs) in the field using MALDI-TOF MS. The legs of 826 mosquitoes were then submitted for MALDI-TOF MS analysis in order to identify the different mosquito species. Eight mosquito species were identified, including Anopheles gambiae Giles, Anopheles coluzzii, Anopheles arabiensis, Culex quinquefasciatus, Culex neavei, Culex perexiguus, Aedes aegypti and Aedes fowleri in Mali. The field mosquitoes for which MALDI-TOF MS did not provide successful identification were not previously available in our database. These specimens were subsequently molecularly identified. The WFP blood meal sources found in this study were matched against human blood (n = 619), chicken blood (n = 9), cow blood (n = 9), donkey blood (n = 6), dog blood (n = 5) and sheep blood (n = 3). This study reinforces the fact that MALDI-TOF MS is a promising tool for entomological surveys.

Accurate identification of Anopheles gambiae Giles trophic preferences by MALDI-TOF MS.

The determination of the trophic preferences of the Anopheles gambiae Giles (Diptera: Culicidae) is a decisive parameter for the monitoring and the prevention of malaria risk transmission. Currently, arthropod blood feeding sources are identified using immunological or molecular biology traditional techniques. Despite the effectiveness of these methods, they present several limitations, and notably, they are time-consuming and costly techniques. A recent study demonstrated that MALDI-TOF MS could be a useful tool for the identification of blood meal origins in freshly engorged mosquitoes. However, the limited number of blood vertebrate species tested to date, did not allow an assessment of the efficiency of MALDI-TOF MS in distinguishing blood MS spectra among close host species, such as humans versus primates. Therefore, in the present study, blood from ten distinct vertebrate host species, including four domestic species, four wild species, and two primates, was selected to control the reliability of MALDI-TOF MS based identification. Host blood species-specific MS profiles, up to 24h post-feeding in engorged Anopheles abdomens, were confirmed. Blind tests underlined the high specificity of MS spectra for the recognition of each host species, preventing misidentification. Nevertheless, an accurate analysis of the results from MS spectra queried against the MS database revealed that the reliability of identification is directly linked to the comprehensiveness of the MS reference database. Finally, the rapidity, the low-cost reagents, the simplicity of data analysis, and the accuracy of the tool for blood origin determination, make this proteomic strategy a promising complementary method for the elucidation of host/vector interactions.

Microbial Culturomics Application for Global Health: Noncontiguous Finished Genome Sequence and Description of Pseudomonas massiliensis Strain CB-1T sp. nov. in Brazil.

Culturomics is a new postgenomics field that explores the microbial diversity of the human gut coupled with taxono-genomic strategy. Culturomics, and the microbiome science more generally, are anticipated to transform global health diagnostics and inform the ways in which gut microbial diversity contributes to human health and disease, and by extension, to personalized medicine. Using culturomics, we report in this study the description of strain CB1T ( = CSUR P1334 = DSM 29075), a new species isolated from a stool specimen from a 37-year-old Brazilian woman. This description includes phenotypic characteristics and complete genome sequence and annotation. Strain CB1T is a gram-negative aerobic and motile bacillus, exhibits neither catalase nor oxidase activities, and presents a 98.3% 16S rRNA sequence similarity with Pseudomonas putida. The 4,723,534 bp long genome contains 4239 protein-coding genes and 74 RNA genes, including 15 rRNA genes (5 16S rRNA, 4 23S rRNA, and 6 5S rRNA) and 59 tRNA genes. Strain CB1T was named Pseudomonas massiliensis sp. nov. and classified into the family Pseudomonadaceae. This study demonstrates the usefulness of microbial culturomics in exploration of human microbiota in diverse geographies and offers new promise for incorporating new omics technologies for innovation in diagnostic medicine and global health.

MALDI-TOF MS identification of Anopheles gambiae Giles blood meal crushed on Whatman filter papers.

BACKGROUND: Identification of the source of mosquito blood meals is an important component for disease control and surveillance. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling has emerged as an effective tool for mosquito blood meal identification, using the abdomens of freshly engorged mosquitoes. In the field, mosquito abdomens are crushed on Whatman filter papers to determine the host feeding patterns by identifying the origin of their blood meals. The aim of this study was to test whether crushing engorged mosquito abdomens on Whatman filter papers was compatible with MALDI-TOF MS for mosquito blood meal identification. Both laboratory reared and field collected mosquitoes were tested. MATERIAL AND METHODS: Sixty Anopheles gambiae Giles were experimentally engorged on the blood of six distinct vertebrate hosts (human, sheep, rabbit, dog, chicken and rat). The engorged mosquito abdomens were crushed on Whatman filter papers for MALDI-TOF MS analysis. 150 Whatman filter papers, with mosquitoes engorged on cow and goat blood, were preserved. A total of 77 engorged mosquito abdomens collected in the Comoros Islands and crushed on Whatman filter papers were tested with MALDI-TOF MS. RESULTS: The MS profiles generated from mosquito engorged abdomens crushed on Whatman filter papers exhibited high reproducibility according to the original host blood. The blood meal host was correctly identified from mosquito abdomens crushed on Whatman filter papers by MALDI-TOF MS. The MS spectra obtained after storage were stable regardless of the room temperature and whether or not they were frozen. The MS profiles were reproducible for up to three months. For the Comoros samples, 70/77 quality MS spectra were obtained and matched with human blood spectra. This was confirmed by molecular tools. CONCLUSION: The results demonstrated that MALDI-TOF MS could identify mosquito blood meals from Whatman filter papers collected in the field during entomological surveys. The application of MALDI-TOF MS has proved to be rapid and successful, making it a new and efficient tool for mosquito-borne disease surveillance.

Use of MALDI-TOF MS and culturomics to identify mosquitoes and their midgut microbiota.

BACKGROUND: Mosquitoes transmit a wide range of human parasitic and viral diseases. In recent years, new techniques such as MALDI-TOF MS have been developed to identify mosquitoes at the species level, which is key for entomological surveys. Additionally, there is increasing interest in the mosquito microbiota and its role in vector capacity. METHODS: The culturomics approach previously used in our laboratory to study human gut microbiota was applied to evaluate the midgut bacterial diversity of Anopheles gambiae (wild and laboratory strains), Aedes albopictus (wild and laboratory strains) and Culex quinquefasciatus (wild strains) in order to determine the influence of the environmental status on the midgut microbiota of the mosquitoes. RESULTS: Mosquitoes collected in the field were accurately identified by MALDI-TOF MS analysis of their legs. Adult mosquito midgut microbiota was composed of four phyla, including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The majority of the bacteria detected in the microbiota of mosquitoes were gram-negative and belong to the phylum Proteobacteria. MALDI-TOF MS identified for the first time a new bacterial species from An. gambiae midgut microbiota. CONCLUSION: In this study, the culturomics approach was found to be a reliable technique for exploring the diversity of the mosquito microbiota. MALDI-TOF MS was confirmed as a promising technique to identify mosquitoes collected in the field. Culturomics allowed the isolation of a new bacterial species not previously associated with mosquito vectors. The environment plays a role in the bacterial diversity of the microbiota, which could enable the development of new control strategies for mosquito-borne disease.