Molecular characterization of misidentified Plasmodium ovale imported cases in Singapore.

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.

[A case of Plasmodium ovale malaria--morphological diagnostic difficulty and utility of rapid diagnostic tests].

A 46-year-old Japanese man was referred to our travel clinic because of high fever for the past 7 days. He worked as an engineer for a month in Zambia and returned to Japan 2 days ago. He had a high-grade fever of 40.5 degrees C. Examination of the palpebral conjunctiva showed no evidence of anemia. Liver and spleen were not palpable. Blood sample was collected at the time of the febrile paroxysm. Malaria parasites were detected by examination of Giemsa-stained thin blood films. The dominant feature of parasite was early trophozoit with a low parasitemia (0.0469%, 1,857.6/microL). The James' stippling was absent. Schizonts and gametocytes were scarce. As ring morphology was quite variable, identification of species might not be possible. Identification of species is more difficult than usual, on the grounds that: 1) the blood sample contains rare early trophozoites, 2) the level of parasitemia is low, and 3) it is quite possible for parasites to be transformed due to the inappropriate treatment. Finally, the diagnosis was confirmed by nested PCR. Examination of Giemsa-stained blood films is the "gold standard" for detection and identification of organisms. However, in non-endemic countries, trained laboratory personnel are scarce and the most may be inexperienced in malaria diagnosis. It is recommended that personnel continue to gain experience by participating in external quality assurance schemes, and that routine laboratories utilize rapid diagnostic tests (RDTs) concurrently. The availability of simple and accurate RDTs could aid the diagnosis in no-endemic countries.

Plasmodium ovale infection in Malaysia: first imported case.

BACKGROUND: Plasmodium ovale infection is rarely reported in Malaysia. This is the first imported case of P. ovale infection in Malaysia which was initially misdiagnosed as Plasmodium vivax. METHODS: Peripheral blood sample was first examined by Giemsa-stained microscopy examination and further confirmed using a patented in-house multiplex PCR followed by sequencing. RESULTS AND DISCUSSION: Initial results from peripheral blood smear examination diagnosed P. vivax infection. However further analysis using a patented in-house multiplex PCR followed by sequencing confirmed the presence of P. ovale. Given that Anopheles maculatus and Anopheles dirus, vectors of P. ovale are found in Malaysia, this finding has significant implication on Malaysia's public health sector. CONCLUSIONS: The current finding should serve as an alert to epidemiologists, clinicians and laboratory technicians in the possibility of finding P. ovale in Malaysia. P. ovale should be considered in the differential diagnosis of imported malaria cases in Malaysia due to the exponential increase in the number of visitors from P. ovale endemic regions and the long latent period of P. ovale. It is also timely that conventional diagnosis of malaria via microscopy should be coupled with more advanced molecular tools for effective diagnosis.