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Malaria is responsible for over 435,000 deaths annually, mostly occurring in sub-Saharan Africa. Detecting Plasmodium spp. sporozoites (spzs) in the salivary glands of Anopheles (Diptera: Culicidae) vectors with circumsporozoite enzyme-linked immunosorbent assay (csELISA) is an important surveillance method. However, current technological advances are intellectual property and often require of distribution and highly trained users. The transition into paper-based rapid plataforms would allow for decentralization of survillance, especially in areas where it was virtually eliminated. The addition of bio-based materials have shown the potential to improve binding of target antigens, while being widely available. Here, we evaluate the use of chitosan and cellulose nanocrystals (CNC) as antibody carriers and substrate coatings on 96-well plates and on wax hydrophobized paper plates for the detection of Plasmodium falciparum (Pf), P. vivax VK210 (Pv210), and P. vivax VK247 (Pv247). To further improve the user-friendliness of the paper plates a quantitative photograph image-based color analysis was done. Interactions between the materials and the assay antibodies were studied by quartz crystal microbalance with dissipation monitoring (QCM-D). Overall, the addition of chitosan increased the interaction with antibodies and enhanced signaling in all tests. This work demonstrated that the adaptation of a PcsELISA shows potential as a cost-effective alternative assay platform easily adaptable in deployable testing sites that also showed reduction in reagent volumes by 80% and assay run time by seventh. While dipstick assays were previously developed, paper-based assays are a cost-effective and field-deployable alternative, reducing volumes of reagents that could be used in malaria control and elimination settings.

BackgroundMalaria is responsible for over 435,000 deaths annually, with most cases occurring in sub-Saharan Africa. Detecting the presence of Plasmodium spp. sporozoites (spzs) in the salivary glands of Anopheles vectors of the parasites using the circumsporozoite enzyme-linked immunosorbent assay (csELISA) is an important malaria surveillance method. The addition of bio-based materials have shown the potential to improve the adsorption and binding of target antigens and thus can improve the sensitivity and detection of analytes in immunoassays. Here, we evaluate the use of two bio-based polymers, chitosan, and cellulose nanocrystals (CNC), as antibody carriers and substrate coating on 96-well plates and on a paper substrate to determine whether detection of Plasmodium falciparum (Pf), P. vivax VK210 (Pv210) and P. vivax VK247 (Pv247) can be improved through assay modification. MethodsModified csELISA assays were conducted using chitosan and CNC as either an antibody carrier or a well coating on 96-well plates (ultra-low- and high-affinity) and results read using standard spectrophotometry of 96-well plates and a quantitative image-based color analysis using photographs of paper plate assays. Changes in frequency and dissipation, resulting from the adsorption of antibodies to model films in a quartz crystal microbalance with dissipation monitoring (QCM-D), were followed to understand better the interactions between the bio-based materials and assay proteins. ResultsThe csELISA performed on high-binding well-plates showed that chitosan, used either as an antibody carrier or well coating, resulted in the greatest increase in detection for Pv210 and Pv247 positive recombinant proteins, increasing absorbance readout values up to 6x for Pv210 and up to 5x for Pv247. On paper csELISA (PcsELISA), chitosan as an antibody carrier yielded the greatest increase in detection sensitivity for all three Plasmodium species when color intensity of positive recombinant proteins was compared to blanks. Compared to controls, chitosan as a carrier resulted in a ~2.5-fold increase in color intensity for Pf, a ~4-fold increase for Pv210, and a ~2-fold increase for Pv247. QCM-D showed a preferred interaction between the assay antibodies and chitosan surfaces, most likely driven by electrostatic interactions. ConclusionThe addition of bio-based materials, mainly chitosan, as shown by QCM-D interactions, absorbance readout values, and image-based color analysis, increased the color intensity of positive samples run through csELISA in all systems, allowing for clearer detection of Plasmodium spp visually, using a spectrophotometer and quantitative color intensity. Furthermore, the adaptation of a PcsELISA coated with chitosan using positive recombinant proteins shows potential as a cost-effective alternative assay platform as it reduced reagent volumes by 80% and assay run time from seven hours to one hour.

Background Malaria is a top cause of mortality on the island nation of Madagascar, where many rural communities rely on subsistence agriculture and livestock production. Understanding feeding behaviours of Anopheles in this landscape is crucial for optimizing malaria control and prevention strategies. Previous studies in southeastern Madagascar have shown that Anopheles mosquitoes are more frequently captured within 50 m of livestock. However, it remains unknown whether these mosquitoes preferentially feed on livestock. Here, mosquito blood meal sources and Plasmodium sporozoite rates were determined to evaluate patterns of feeding behaviour in Anopheles spp. and malaria transmission in southeastern Madagascar. Methods Across a habitat gradient in southeastern Madagascar 7762 female Anopheles spp. mosquitoes were collected. Of the captured mosquitoes, 492 were visibly blood fed and morphologically identifiable, and a direct enzyme-linked immunosorbent assay (ELISA) was used to test for swine, cattle, chicken, human, and dog blood among these specimens. Host species identification was confirmed for multiple blood meals using PCR along with Sanger sequencing. Additionally, 1,607 Anopheles spp. were screened for the presence of Plasmodium falciparum, P. vivax-210, and P. vivax 247 circumsporozoites (cs) by ELISA. Results Cattle and swine accounted, respectively, for 51% and 41% of all blood meals, with the remaining 8% split between domesticated animals and humans. Of the 1,607 Anopheles spp. screened for Plasmodium falciparum, Plasmodium vivax 210, and Plasmodium vivax 247 cs-protein, 45 tested positive, the most prevalent being P. vivax 247, followed by P. vivax 210 and P. falciparum. Both variants of P. vivax were observed in secondary vectors, including Anopheles squamosus/cydippis, Anopheles coustani, and unknown Anopheles spp. Furthermore, evidence of coinfection of P. falciparum and P. vivax 210 in Anopheles gambiae sensu lato (s.l.) was found. Conclusions Here, feeding behaviour of Anopheles spp. mosquitoes in southeastern Madagascar was evaluated, in a livestock rich landscape. These findings suggest largely zoophagic feeding behaviors of Anopheles spp., including An. gambiae s.l. and presence of both P. vivax and P. falciparum sporozoites in Anopheles spp. A discordance between P. vivax reports in mosquitoes and humans exists, suggesting high prevalence of P. vivax circulating in vectors in the ecosystem despite low reports of clinical vivax malaria in humans in Madagascar. Vector surveillance of P. vivax may be relevant to malaria control and elimination efforts in Madagascar. At present, the high proportion of livestock blood meals in Madagascar may play a role in buffering (zooprophylaxis) or amplifying (zoopotentiation) the impacts of malaria. With malaria vector control efforts focused on indoor feeding behaviours, complementary approaches, such as endectocide-aided vector control in livestock may be an effective strategy for malaria reduction in Madagascar.

Background When rearing morphologically indistinguishable laboratory strains concurrently, the threat of unintentional genetic contamination is constant. Avoidance of accidental mixing of strains is difficult due to the use of common equipment, technician error, or the possibility of self relocation by adult mosquitoes ("free fliers"). In many cases, laboratory strains are difficult to distinguish because of morphological and genetic similarity, especially when laboratory colonies are isolates of certain traits from the same parental strain, such as eye color mutants, individuals with certain chromosomal arrangements or high levels of insecticide resistance. Thus, proving genetic integrity could seem incredibly time-consuming or impossible. On the other hand, lacking proof of genetically isolated laboratory strains could question the validity of research results. Results We present a method for establishing authentication matrices to routinely distinguish and confirm that laboratory strains have not become physically or genetically mixed through contamination events in the laboratory. We show a specific example with application to Anopheles gambiae sensu stricto strains at the Malaria Research and Reference Reagent Resource Center. This authentication matrix is essentially a series of tests yielding a strain-specific combination of results. Conclusion These matrix-based methodologies are useful for several mosquito and insect populations but must be specifically tailored and altered for each laboratory based on the potential contaminants available at any given time. The desired resulting authentication plan would utilize the least amount of routine effort possible while ensuring the integrity of the strains.