Multiple genotype and multi-species infections of malaria (and other pathogens) are extremely common. For example >80% of human P. falciparum infections in sub-Saharan Africa contain multiple parasite genotypes. Most malaria species cannot be cultured in the laboratory, precluding culture based isolation of individual clones. Currently, genetic analysis of such complex infections involves either characterization of hypervariable genome regions or more recently, deep sequencing of infections. Both methods are inadequate because parasite haplotypes cannot be constructed and statistical methods for resolving complex haplotype mixtures are not yet available. To resolve this problem, we have developed robust methods for single cell genomics of Plasmodium, utilizing FACs to isolates single infected cells, followed by whole genome amplification, and genetic characterization and/or deep sequencing. These methods open up new opportunities for understanding the composition of complex malaria infections, for validating statistical methods that seek to reconstruct haplotypes from deep sequence data, and to examine the within host-dynamics of non-culturable malaria infections. We exploit these new methods: (1) to dissect the composition of P. falciparum infections in a region of intense transmission. These data will be used to determine how many independent genotypes are present, the relationships between the component haplotypes and their relative abundance within infections, and to critically evaluate the performance of indirect methods currently employed. (2) We will dissect complex P. vivax infections in Thai patients in primary and subsequent infections. This work is not possible through other means as unlike P. falciparum no long term culture is possible for P. vivax. We will clarify the relationships between primary and subsequent infections, and within host dynamics of this important human pathogen.
Infections with malaria parasites frequently contain both multiple Plasmodium species and multiple haplotypes from each species. Multiple species and haplotype infections impact disease severity, the spread of drug resistance and basic population genetic parameters, and complicate even basic genetic analysis. This study applies novel approaches developed in our laboratory which address this problem by generating whole genome sequence from single parasite-infected cells. We will apply these to human infections to refine our understanding of the complexity of malaria infections.
| Trevino, Simon G; Nkhoma, Standwell C; Nair, Shalini et al. (2017) High-Resolution Single-Cell Sequencing of Malaria Parasites. Genome Biol Evol 9:3373-3383 |
