Molecular characterization of misidentified Plasmodium ovale imported cases in Singapore

Abstract

BackgroundPlasmodium 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.MethodsMorphological 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).ResultsThe 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.ConclusionMisidentified 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0985-8) contains supplementary material, which is available to authorized users.

Parasitological correlates of Plasmodium ovale curtisi and Plasmodium ovale wallikeri infection

“…Among peptide sequences of DHFR-TS in isolates of P. ovale curtisi and P. ovale wallikeri studied herein, mutations known to confer antifolate resistance in other species of malaria, notably P. falciparum were not demonstrated Furthermore, non-synonymous mutations within each sub-species, P. ovale wallikeri in particular, were also observed, confirming previous reports [2]. While there are variations in the peptide sequences between sub-species, it is unknown how these polymorphisms influence enzyme activity.…”

“…Several differences in the peptide sequences between P. ovale curtisi and P. ovale wallikeri were observed (Table 3). Non-synonymous mutations were observed within the P. ovale wallikeri isolates as previously reported [2]. Specifically, deletions of two or four amino acids (i.e., TA252–253 or TATA252–255) were identified in six and one P. ovale wallikeri isolates, respectively (Table 3).…”

“…Alignments of P. ovale sequences were performed using Mega6 software [17]. Genetic and peptide sequences between the enrolled specimens and complete P. ovale sequences that were previously reported (GenBank: EU266601, EU266604–EU266618, GQ250090, GQ250091, KP050409, and KP05413–KP050415) [2, 7, 18] were compared.…”

“…Prevalent in West and East Africa and the Asia–Pacific [1], P. ovale infections can be challenging to diagnose in part due to the typical presentation of clinically mild disease and low parasite burden [2]. Consequently, conventional diagnostic tools, such as microscopy of blood films and rapid diagnostic tests (RDT), have limited performance in detecting P. ovale infection [3–5], often resulting in the use of molecular techniques to confirm the diagnosis [5].…”

“…It is known that the dihydrofolate reductase-thymidylate synthase ( dhfr - ts ) domain, a common anti-malarial drug target and a region that can be linked to antifolate resistance, has been thoroughly investigated in Plasmodium falciparum and Plasmodium vivax . However, investigations into this region have only recently begun for P. ovale isolates [2, 8, 11]. Previous reports have implicated specific sites in the peptide sequence that may interfere with antifolate binding at the active site, which include D53, F57, S58, T61, S113, S116, I169, and T190 [8, 11].…”