Molecular characterization of misidentified Plasmodium ovale imported cases in Singapore

Abstract

Background: Plasmodium ovale, considered the rarest of the malaria parasites of humans, consists of two morphologically identical but genetically distinct sympatric species, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. These parasites resemble morphologically to Plasmodium vivax with which they also share a tertian periodicity and the ability to cause relapses, making them easily misidentified as P. vivax. Plasmodium ovale infections are rarely reported, but given the likelihood of misidentification, their prevalence might be underestimated.

Methods: Morphological and molecular analysis of confirmed malaria cases admitted in Singapore in 2012-2014 detected nine imported P. ovale cases that had been misidentified as P. vivax. Since P. ovale had not been previously officially reported in Singapore, a retrospective analysis of available, frozen, archival blood samples was performed and returned two additional misidentified P. ovale cases in 2003 and 2006. These eleven P. ovale samples were characterized with respect to seven molecular markers (ssrRNA, Potra, Porbp2, Pog3p, dhfr-ts, cytb, cox1) used in recent studies to distinguish between the two sympatric species, and to a further three genes (tufa, clpC and asl).

Results: The morphological features of P. ovale and the differential diagnosis with P. vivax were reviewed and illustrated by microphotographs. The genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was assessed by ten molecular markers distributed across the three genomes of the parasite (Genbank KP050361-KP050470). The data obtained for seven of these markers were compared with those published and confirmed that both P. ovale species were present. This dimorphism was also confirmed for the first time on: (1) two genes from the apicoplast genome (tufA and clpC genes); and, (2) the asl gene that was used for phylogenetic analyses of other Plasmodium species, and that was found to harbour the highest number of dimorphic loci between the two P. ovale species.

Conclusion: Misidentified P. ovale infections are reported for the first time among imported malaria cases in Singapore. Genetic dimorphism between P. ovale curtisi and P. ovale wallikeri was confirmed using markers from the parasites' three genomes. The apparent increase of imported P. ovale since 2012 (with yearly detection of cases) is puzzling. Given decrease in the overall number of malaria cases recorded in Singapore since 2010 the 'resurgence' of this neglected species raises public health concerns.

Fig. 1

Microphotographs of Plasmodium ovale in Giemsa-stained thin blood films collected in Singapore. a–c ring stages, d, e young trophozoites, f trophozoite, g late trophozoite, h young schizont, i–k growing schizont, l late schizont, m ruptured schizont, n young gametocyte, o, p developing macrogametocytes, q macrogametocyte, r microgametocyte. a–e, h–m, o, q and r are P. ovale curtisi while f, g, n and p are P. ovale wallikeri

Fig. 2

Microphotographs of Plasmodium vivax in Giemsa-stained thin blood films collected in Singapore. a, b ring stages, c–e young trophozoites, f–h amoeboid trophozoites, i young schizont, j–l growing schizonts, m developed schizont, n mature schizont, o young gametocyte, p macrogametocyte, r, q microgametocytes

Fig. 3

Dimorphic nature of Plasmodium ovale curtisi and Plasmodium ovale wallikeri based on the partial sequences from three nuclear and two mitochondrial genes. a 18S ssrRNA, b Porbp2, c Pog3p, d cox1, e cytb genes. Each alignment includes sequences from the P. ovale cases imported in Singapore (highlighted in grey) and from published sequences. Numbering is based for the 18S ssrRNA gene on the asexually expressed (A type) gene [L48987], for the Porbp2 and Pog3p genes on the putative start-codons (ATG) identified by [23], for the cox1 gene on the homologous position of the P. falciparum cox1 gene [M76611], and for the cytb gene on the start-codon (ATG). For 18S ssrRNA gene the sets of sequences A1 and A2 contain: [L48986, L48987, AB182489, GQ231515, GQ183065, GQ183068] and [GU813972, JF894422, JF894425, JF894426] respectively; for Porbp2 gene the sets of sequences B1 and B2 contain: [GU813972, JF894422, JF894425, JF894426] and [GU813971, JF894427-JF894429] respectively; for cytb gene the sets of sequences E1 to E4 gene contain [HQ712053, GQ231518-GQ231520], [GU723535-GU723537, GU723539-GU723548], [HQ712052, AB354571, GQ231516-GQ231517] and [AB182496, AF069625, GU723514-GU723534], respectively. The Singapore P. o. wallikeri and Singapore P. o. curtisi sets represent for each gene the sequences obtained from [Po 2006, Po 2012-3 and Po-2014-2] and from [Po 2003, Po 2012-1, Po 2012-2, Po 2012-4, Po 2013-1, Po 2013-2, Po 2013-3 and Po-2014-1], respectively. Non-synonymous mutations are in boldface type, hyphens represent gaps, dots represent nucleic acid identity and blanks represent unavailable information

Fig. 4

Dimorphic nature of Plasmodium ovale curitsi and Plasmodium ovale wallikeri based on the translated amino acid sequences from two additional nuclear genes. a Potra gene, and b dhfr-ts gene. Each alignment includes sequences from the imported P. ovale cases imported in Singapore (highlighted in grey) and from published sequences. Dimorphic sites are in boldface type. For Potra gene, the distinction of between P. o. wallikeri types 1 and 2 is based on a two amino acid variation located outside of the amplified fragment presented here [24], and the distinction between P. o. curtisi types 1 and 2 is based on the number of PISTIT repeat (n = 1 or 2) [23, 24]. For dhfr-ts gene, in addition to the dimorphisms between P. o. wallikeri and P. o. curtisi, the amino acid polymorphisms within each species are included to illustrate internal variations. In Potra gene for P. o. wallikeri the green and blue boxes represent the PINMAN repeats (n = 1–5) region and the PINAIT repeats (n = 2–4) region, respectively; for P. o. curtisi the orange box represents the PISTIT repeat (n = 1 or 2) region. In dhfr-ts the red box represents the AT repeats (n = 1 or 2) region. Numbering is based on the ORF identified by [24] for Potra, and on the putative start-codon (ATG) of dhfr-ts. Hyphens represent gaps and dots represent amino acid identity

Fig. 5

Combined phylogenetic trees generated by maximum likelihood (ML) (left part) and neighbour joining (NJ) (right part) methods using the partial sequences of the asl gene from different Plasmodium species. Of the sequences in the dataset, the 15 obtained in the present study are in boldface type (cases imported in Singapore: 8 P. o. curtisi (dark green box), 3 P. o. wallikeri (light green box), with 1 P. vivax, 1 P. falciparum, 1 P. knowlesi, and 1 P. malariae), the other 15 Plasmodium sequences (GenBank accession number is provided between vertical bars) from Plasmodium species that infect humans and primates (P. falciparum and Plasmodium reichenowi, red box; P. malariae, yellow box; Plasmodium fragile, Plasmodium simiovale, Plasmodium inui, Plasmodium cynomolgi, P. knowlesi and P. vivax, blue box), apes, rodents (Plasmodium yoelii, Plasmodium berghei, Plasmodium vinckei, and Plasmodium chabaudi, purple box) and birds (Plasmodium gallinaceum, orange box). The homologous fragment from the Toxoplasma gondii asl gene was used at the out-group to root the trees. After alignment the sequences have been trimmed (≈800 bp) to adjust their size to the fragment with the most coverage

Fig. 6

Dimorphic nature of Plasmodium ovale curtisi and Plasmodium ovale wallikeri based on the partial sequences from the nuclear gene asl. The alignment highlights differences between P. o. curtisi on P. o. wallikeri at 62 loci. Numbering is based on the first nucleic acid of the forward oligonucleotide primer. Non-synonymous mutations are in boldface type, and dots represent nucleic acid identity

Fig. 7

Dimorphic nature of Plasmodium ovale curtisi and Plasmodium ovale wallikeri based on the partial sequences from two apicoplast genes. a tufA gene, and b clpC gene. Each alignment includes the sequences from the P. ovale cases imported in Singapore (highlighted in grey) and published sequences. The Plasmodium ovale-like sequence [HQ842632] isolated from chimpanzee (Kaiser et al. unpublished) is included into this comparison but separated from P. o. curtisi and P. o wallikeri because it present 11 additional unique polymorphic loci beside the 15 dimorphic loci that distinguish P. o. curtisi and P. o wallikeri. Numbering is based on the start-codon (ATG) identified for both tufA and clpC. Non-synonymous mutations are in boldface type and dots represent nucleic acid similarity