Use of qPCR and genomic sequencing to diagnose Plasmodium ovale wallikeri malaria in a returned soldier in the setting of a negative rapid diagnostic assay

Abstract

Plasmodium ovale is one of several clinically relevant malaria species known to cause disease in humans. However, in contrast to Plasmodium falciparum and Plasmodium vivax, which are responsible for most cases of human malaria, P. ovale has a wide distribution but low prevalence in tropical regions. Here, we report the case of a soldier returning from Liberia with P. ovale wallikeri malaria. This case highlights the limitations of both microscopy and the malaria rapid diagnostic test for diagnosing infection with P. ovale and for distinguishing P. ovale wallikeri from P. ovale curtisi. To our knowledge, this is the first case report in which quantitative real-time polymerase chain reaction amplification using the Cytochrome B gene, coupled with genomic sequencing of the potra locus, was used for definitive diagnosis of P. ovale wallikeri malaria.

Figure 1.

Thick peripheral blood smears showing intra-erythrocytic structures consistent with malaria (1,000×).

Figure 2.

Alignment of Plasmodium ovale tra gene sequences. Nucleotide sequences were aligned and compared using ClustalW alignment program in Geneious Pro v5.6.4. Five partial tra sequences were used in the analysis (from top): P. ovale wallikeri type 1 tra gene (HM594180), P. ovale wallikeri type 2 tra gene (HM594181), P. ovale curtisi type 1 tra gene (HM594182), P. ovale curtisi type 2 tra gene (HM594183), and the Potra gene sequenced in this study. The numbers for nucleotide positions are shown above the alignment graph. Each vertical line indicates a nucleotide that is different from the other sequences. Sequence comparison for the variable region is shown in the box. Nucleotides different from the other sequences, i.e., corresponding to the vertical lines in the alignment graph are shown in red.

Figure 3.

Thin smear showing structures suggestive of Plasmodium ovale malaria. (A) Enlarged “comet” cell (an elongated cell with fimbriae resembling a comet); (B) oval cell with fimbriae; (C) enlarged oval cell with fimbriae; (D) enlarged oval cell with rough edge and James' dots.

Figure 4.

Thin smear showing structures atypical for Plasmodium ovale malaria. (A–E) Infected cells that are smaller than uninfected cells, suggestive of P. malariae; (F) ameboid cell and cytoplasm suggestive of P. vivax.