Novel highly-multiplexed AmpliSeq targeted assay for Plasmodium vivax genetic surveillance use cases at multiple geographical scales.

Although the power of genetic surveillance tools has been acknowledged widely, there is an urgent need in malaria endemic countries for feasible and cost-effective tools to implement in national malaria control programs (NMCPs) that can generate evidence to guide malaria control and elimination strategies, especially in the case of Plasmodium vivax. Several genetic surveillance applications ('use cases') have been identified to align research, technology development, and public health efforts, requiring different types of molecular markers. Here we present a new highly-multiplexed deep sequencing assay (Pv AmpliSeq). The assay targets the 33-SNP vivaxGEN-geo panel for country-level classification, and a newly designed 42-SNP within-country barcode for analysis of parasite dynamics in Vietnam and 11 putative drug resistance genes in a highly multiplexed NGS protocol with easy workflow, applicable for many different genetic surveillance use cases. The Pv AmpliSeq assay was validated using: 1) isolates from travelers and migrants in Belgium, and 2) routine collections of the national malaria control program at sentinel sites in Vietnam. The assay targets 229 amplicons and achieved a high depth of coverage (mean 595.7 ± 481) and high accuracy (mean error-rate of 0.013 ± 0.007). P. vivax parasites could be characterized from dried blood spots with a minimum of 5 parasites/µL and 10% of minority-clones. The assay achieved good spatial specificity for between-country prediction of origin using the 33-SNP vivaxGEN-geo panel that targets rare alleles specific for certain countries and regions. A high resolution for within-country diversity in Vietnam was achieved using the designed 42-SNP within-country barcode that targets common alleles (median MAF 0.34, range 0.01-0.49. Many variants were detected in (putative) drug resistance genes, with different predominant haplotypes in the pvmdr1 and pvcrt genes in different provinces in Vietnam. The capacity of the assay for high resolution identity-by-descent (IBD) analysis was demonstrated and identified a high rate of shared ancestry within Gia Lai Province in the Central Highlands of Vietnam, as well as between the coastal province of Binh Thuan and Lam Dong. Our approach performed well in geographically differentiating isolates at multiple spatial scales, detecting variants in putative resistance genes, and can be easily adjusted to suit the needs in other settings in a country or region. We prioritize making this tool available to researchers and NMCPs in endemic countries to increase ownership and ensure data usage for decision-making and malaria policy.

Colorimetric Detection of Carcinogenic Alkylating Fumigants on a Nylon 6 Nanofibrous Membrane. Part II: Self-Catalysis of 2-Diethylaminoethyl-Modified Sensor Matrix for Improvement of Sensitivity.

A nylon 6 nanofibrous membrane (N6NFM) was covalently modified with 2-diethylaminoethylchloride (DEAE-Cl) to provide self-catalytic functions to facilitate the formation of color compounds in reactions of 4-( p-nitrobenzyl)pyridine with alkylating fumigants. The 2-diethylaminoethyl group on the DEAE-Cl-modified N6NFM (DEAE@N6NFM) enables effective elimination of hydrohalogenic acids from intermediates that were formed from reactions between the alkylating fumigants and NBP and consequently improve their detection sensitivities, especially for 1,3-dichloropropene at room temperature. Moreover, DEAE@N6NFM can be recycled and reused multiple times without obvious loss in the sensing functions or any noticeable material damage. The naked-eye detection limits of the sensor to 1,3-dichloropropene, methyl iodide, and methyl bromide on DEAE@N6NFM are improved to 0.2, 0.1, and 0.1 ppm, respectively, which are much lower than their occupational exposure limits. The reaction mechanism is demonstrated through a computational method by analyzing the thermodynamics of the reaction. The modification of DEAE@N6NFM also provides an insight into the development of functionalized materials with improved reactivities for versatile sensing applications.

Partial mitochondrial DNA sequences suggest the existence of a cryptic species within the Leucosphyrus group of the genus Anopheles (Diptera: Culicidae), forest malaria vectors, in northern Vietnam.

BACKGROUND: During the last decade, Southeast Asian countries have been very successful in reducing the burden of malaria. However, malaria remains endemic in these countries, especially in remote and forested areas. The Leucosphyrus group of the genus Anopheles harbors the most important malaria vectors in forested areas of Southeast Asia. In Vietnam, previous molecular studies have resulted in the identification of only Anopheles dirus sensu stricto (previously known as An. dirus species A) among the Leucosphyrus group members. However, Vietnamese entomologists have recognized that mosquitoes belonging to the Leucosphyrus group in northern Vietnam exhibit morphological characteristics similar to those of Anopheles takasagoensis, which has been reported only from Taiwan. Here, we aimed to confirm the genetic and morphological identities of the members of the Leucosphyrus group in Vietnam. RESULTS: In the molecular phylogenetic trees reconstructed using partial COI and ND6 mitochondrial gene sequences, samples collected from southern and central Vietnam clustered together with GenBank sequences of An. dirus that were obtained from Thailand. However, samples from northern Vietnam formed a distinct clade separated from both An. dirus and An. takasagoensis by other valid species. CONCLUSIONS: The results suggest the existence of a cryptic species in northern Vietnam that is morphologically similar to, but phylogenetically distant from both An. dirus and An. takasagoensis. We have tentatively designated this possible cryptic species as Anopheles aff. takasagoensis for convenience, until a valid name is assigned. However, it is difficult to distinguish the species solely on the basis of morphological characteristics. Further studies on such as karyotypes and polytene chromosome banding patterns are necessary to confirm whether An. aff. takasagoensis is a valid species. Moreover, studies on (1) the geographic distribution, which is potentially spreading along the Vietnam, China, Laos, and Myanmar borders; (2) morphological and ecological characteristics; and (3) vectorial capacity of this newly identified cryptic species of An. dirus, which is one of the most important malaria vectors in the mainland of Southeast Asia, are necessary for planning efficient malaria vector control programs in this region.