PMC4704402_01
Microphotographs of giemsa-stained thin blood films of the patient viewed under 1000 x  magnification. a Developing schizont. b A schizont with eight merozoites. c, d Gametocyte. Infected red blood cell is enlarged, oval in shape with presence of Schuffner's dots, clearly seen in b and d
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Fig1
Malaria Test (USA), a T2 Line appeared, while no T1 Line (specific for P. falciparum infection) was observed, suggesting vivax, malariae or ovale malaria or a mix of these. Microscopy on thick and thin blood films detected the presence of malaria parasites suggesting P. ovale or P. vivax (Fig. 1), with 0.27 % parasitaemia.
2 % agarose gel electrophoresis of Nest 2 PCR showing an amplified product with the genus-specific primers. 1 100 bp DNA ladder; 2 rFAL1/rFAL2 (P. falciparum); 3 rOVA1/rOVA2 (P. ovale); 4 rMAL1/rMAL2 (P. malariae); 5 rVIV1/rVIV2 (P. vivax); 6 Pmk8/Pmkr9 (P. knowlesi); 7 rPLU3/rPLU4 (Plasmodium spp.); 8-13 no template control of P. falciparum, P. ovale, P. malariae, P. vivax, P. knowlesi, and Plasmodium spp., respectively
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Fig2
Gel electrophoresis showed a band of 240 bp, corresponding to the product of the genus-specific nest 2 PCR (Fig. 2).
Phylogenetic analysis of the NG-PCR product (UM00134). The phylogenetic tree is constructed using the Neighbor-Joining methods and Jukes-Cantor model, with Bootstrap value of 1000. The patient isolate, UM00134 is clustered with P. ovale wallikeri isolates. GenBank accession number is given after each isolate's name
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Fig3
A phylogenetic tree was constructed using the neighbour-joining methods and Jukes-Cantor model, with a Bootstrap value of 1000 (Fig. 3).
Detection of P. ovale wallikeri using primer sets rOVA1/rOVA2, rOVA1v/rOVA2v, mixture of both primer sets and rOVA1WC/rOVA2WC. A 2 % agarose gel electrophoresis was performed. Parasite DNA purified from patient blood is used as template of PCR. 1 100 bp DNA ladder; 2 rOVA1/rOVA2 (P. ovale curtisi); 3 rOVA1v/rOVA2v (P. ovale wallikeri); 4 Mixture of primer sets rOVA1/rOVA2 and rOVA1v/rOVA2v (both forms of P. ovale); 5 rOVA1WC/rOVA2WC (both forms of P. ovale)
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Fig4
The mixture of these primer sets in a single Nest 2 PCR reaction and primer set rOVA1WC/rOVA2WC were able to detect P. ovale wallikeri (Fig. 4).