Species and genotype diversity of Plasmodium in malaria patients from Gabon analysed by next generation sequencing

Abstract

Background: Six Plasmodium species are known to naturally infect humans. Mixed species infections occur regularly but morphological discrimination by microscopy is difficult and multiplicity of infection (MOI) can only be evaluated by molecular methods. This study investigated the complexity of Plasmodium infections in patients treated for microscopically detected non-falciparum or mixed species malaria in Gabon.

Methods: Ultra-deep sequencing of nucleus (18S rRNA), mitochondrion, and apicoplast encoded genes was used to evaluate Plasmodium species diversity and MOI in 46 symptomatic Gabonese patients with microscopically diagnosed non-falciparum or mixed species malaria.

Results: Deep sequencing revealed a large complexity of confections in patients with uncomplicated malaria, both on species and genotype levels. Mixed infections involved up to four parasite species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale curtisi, and P. ovale wallikeri). Multiple genotypes from each species were determined from the asexual 18S rRNA gene. 17 of 46 samples (37%) harboured multiple genotypes of at least one Plasmodium species. The number of genotypes per sample (MOI) was highest in P. malariae (n = 4), followed by P. ovale curtisi (n = 3), P. ovale wallikeri (n = 3), and P. falciparum (n = 2). The highest combined genotype complexity in samples that contained mixed-species infections was seven.

Conclusions: Ultra-deep sequencing showed an unexpected breadth of Plasmodium species and within species diversity in clinical samples. MOI of P. ovale curtisi, P. ovale wallikeri and P. malariae infections were higher than anticipated and contribute significantly to the burden of malaria in Gabon.

Keywords: Amplicon; Metagenomics; Next-generation sequencing; Plasmodium falciparum; Plasmodium malariae; Plasmodium ovale curtisi; Plasmodium ovale wallikeri.

Fig. 1

Bioinformatics pipeline. Flowchart describing bioinformatic pipeline. The pipeline is divided into a quality control part, mapping and variant calling part. * used with default settings

Fig. 2

Schematic representation of the sequencing approach by Sanger method to determine the new 18S rRNA gene type sequence of P. ovale spp. Conserved pan-Plasmodium 18S primers (rPLU6 and rPLU5) were first used to amplify the gene spanning three variable regions and two conserved blocks. The nucleotide sequences upstream and downstream of the 454 sequencing target region (V5) of the gene were determined by direct sequencing of the initial product using p-AVL-F and p-AVL-R primers. Partial 18S gene (1078 bp length) was obtained by assembling 454 reads with the sequences obtained using p-AVL-F and p-AVL-R primers

Fig. 3

Relative frequency of each Plasmodium spp. genotype within one infected host. a Relative frequency of P. falciparum genotypes (defined by the 18S A-type gene variation); b relative frequency of P. malariae genotypes. c Relative frequency of P. ovale curtisi and P. ovale wallikeri