Understanding Plasmodium vivax transmission networks through a novel microhaplotype and kinship analysis approach

Project Summary When transmission decreases faster than a population loses immunity, subclinical or asymptomatic carriers remain and can serve as a reservoir for forward transmission. Thus, reactive case detection (RCD), the following up of individuals who reside nearby an index case, is being considered or is already implemented as a malaria intervention in low transmission areas to detect and treat asymptomatic infections. RCD is rooted in the observation that Plasmodium spp. infections tend to distribute in “hotspots” or spatially and temporally related clusters. However, the contribution of imported infections, likely resulting from human travel, or relapse infections to malaria transmission compromises the effectiveness of RCD as a malaria control tool in low transmission areas. Transmission network analysis with high resolution genomic data can be used to assess the effectiveness of RCD as an intervention strategy. Highly multiplexed amplicon sequencing (HM-AmpSeq) of highly polymorphic microhaplotype markers is one powerful solution which could provide the genomic resolution needed for transmission network analysis, while also being low cost, highly sensitive to detect minor infections, and allows us to genotype point mutations of interest (e.g. drug tolerance markers) and resolve multilocus haplotypes in complex infections under certain conditions. Transmission network analysis for Plasmodium spp. has been hindered in part by genetic diversity being primarily driven by recombination coupled with common inbreeding due to the closed mating environment within the vector, which alters expected kinship coefficients that vary by epidemiological context. A novel approach to overcome these barriers is to generate known pedigrees using genetic-epidemiologic simulations, which are used to accommodate uncertainty for kinship analysis. We have developed a preliminary genome-wide P. vivax highly multiplexed microhaplotype panel using global genomic data that has been successfully applied to dried blood spot samples with parasite densities as low as 32 parasites/µl. In Aim 1, we propose to further optimize this protocol to sequence samples with even lower parasite densities by using promising enrichment strategies and strategically down selecting low performing markers. In Aim 2, we will generate known pedigrees through genetic-epidemiologic simulations and apply the microhaplotype panel and kinship analysis approach based on the likelihood ratio test statistic to P. vivax samples collected in an RCD study to determine transmission networks. The overall objective of this project is to develop an optimized genotyping and analytical method for reconstructing transmission chains of P. vivax and apply the method to determine P. vivax transmission networks in Ethiopia. The novel approach developed through this project could be broadly applied to improve our understanding of transmission networks for assessing the effectiveness of potential malaria interventions.