Abstract
The genomic diversity of Plasmodium malariae malaria parasites is understudied, partly because infected individuals tend to present with low parasite densities, leading to difficulties in obtaining sufficient parasite DNA for genome analysis. Selective whole genome amplification (SWGA) increases the relative levels of pathogen DNA in a clinical sample, but has not been adapted for P. malariae parasites. Here we design customized SWGA primers which successfully amplify P. malariae DNA extracted directly from unprocessed clinical blood samples obtained from patients with P. malariae-mono-infections from six countries, and further test the efficacy of SWGA on mixed infections with other Plasmodium spp. SWGA enables the successful whole genome sequencing of samples with low parasite density (i.e. one sample with a parasitaemia of 0.0064% resulted in 44% of the genome covered by ≥ 5 reads), leading to an average 14-fold increase in genome coverage when compared to unamplified samples. We identify a total of 868,476 genome-wide SNPs, of which 194,709 are unique across 18 high-quality isolates. After exclusion of the hypervariable subtelomeric regions, a high-quality core subset of 29,899 unique SNPs is defined. Population genetic analysis suggests that P. malariae parasites display clear geographical separation by continent. Further, SWGA successfully amplifies genetic regions of interest such as orthologs of P. falciparum drug resistance-associated loci (Pfdhfr, Pfdhps, Pfcrt, Pfk13 and Pfmdr1), and several non-synonymous SNPs were detected in these genes. In conclusion, we have established a robust SWGA approach that can assist whole genome sequencing of P. malariae, and thereby facilitate the implementation of much-needed large-scale multi-population genomic studies of this neglected malaria parasite. As demonstrated in other Plasmodia, such genetic diversity studies can provide insights into the biology underlying the disease and inform malaria surveillance and control measures.
Highlights
Molecular surveillance has demonstrated that non-falciparum malaria has been underestimated by microscopy diagnosis[2,3,4,5], and rapid diagnostic tests (RDT), which are unable to diagnose non-falciparum malaria to the species level[6,7]
P. malariae parasites are commonly subject to antimalarial treatments, we investigated the coverage and prevalence of mutations in orthologs of known P. falciparum genes associated with drug resistance (Pfcrt, Pfdhfr, Pfdhps, Pfk[13] and Pfmdr[1]; gene IDs are in S5 Table)
Genetic investigation of this parasite may allow us to understand how P. malariae is able to cause chronic infections, why there are accounts of P. malariae parasites persisting after treatment with Artemisinin Combination Therapy (ACT), and why some P. malariae infections lead to severe outcomes whilst others remain asymptomatic
Summary
Molecular surveillance has demonstrated that non-falciparum malaria has been underestimated by microscopy diagnosis[2,3,4,5], and rapid diagnostic tests (RDT), which are unable to diagnose non-falciparum malaria to the species level[6,7]. WGS data for Plasmodium parasites has been obtained using DNA extracted from venous blood of clinical cases that were pre-filtered to remove human leukocytes, in order to reduce the amount of co-extracted human DNA30 This methodology is efficient when parasite densities are high, this is not the case for the majority of P. malariae infections, asymptomatic individuals, where this approach would not yield sufficient parasite DNA for WGS. After selecting 18 high quality samples, we demonstrate that the resulting WGS data can be used to assess genetic diversity in P. malariae genes orthologous to known drug resistance markers in other species, and to inform population structure. We provide proof-of-principle for large-scale WGS studies using blood samples collected from malaria endemic regions to inform malaria control efforts, and provide new molecular information for development of diagnostics, vaccines and drugs
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