Diagnosis of P. ovale is usually made by the examination of peripheral blood films stained with Giemsa stain. Differentiation from the human malaria parasite, P. vivax, is most difficult. A detailed comparison was made by Wilcox et al. (97) of two strains of P. vivax (Chesson and St. Elizabeth) and the Donaldson strain of P. ovale. Cellular enlargement is a characteristic of both species. Erythrocytes containing ring stages of Chesson and St. Elizabeth showed enlargements of 10.3 and 11.5%, respectively, whereas in the Donaldson strain parasitized cells were the same as uninfected cells. With the binucleate schizont, the Chesson increased in size by 52.9%, the St. Elizabeth by 44.0%, and the Donaldson by 26.8%. Erythrocytes containing mature schizonts increased in size for Chesson 55.8%, for St. Elizabeth 50.7%, and for Donaldson 27.4%. The average number of merozoites for Chesson was 17.3, for St. Elizabeth 14.1, and for Donaldson 7.8. Thus, in comparison with P. vivax, P. ovale does not enlarge the infected erythrocyte as much and produces much fewer merozoites.
About 20% of erythrocytes infected with P. vivax were elliptical, with 2% definitely elongated. In contrast, 35% of P. ovale-infected erythrocytes were elliptical and 16% had a definitely long, narrow, oval or otherwise elongated form. When ring-infected erythrocytes were examined for the presence of Schüffner's stippling, it was much more numerous in P. ovale than in either strain of P. vivax.
As described by Coatney et al. (20) (Fig. 3), the young ring forms of P. ovale have a prominent circular nucleus with a wisp of cytoplasm. As the parasite grows, the erythrocyte becomes enlarged; older trophozoites occupy about half the erythrocyte. The host cell may appear oval with fimbriated edges. This is especially marked in erythrocytes of splenectomized chimpanzees infected with P. ovale (Fig. 4). The pigment is initially in the form of dust-like grains that later come together to form greenish-brown beads; eventually they mass together in yellowish-brown patches. The most distinctive characteristic is the stippling. This appears early and becomes intense as the parasite develops. The stippling is more intense than that of P. vivax.
Gametocytes grow to fill the enlarged host cell. The macrogametocyte stains blue with Giemsa. The pigment is in granules arranged like a string of beads. Stippling is prominent and is arranged in a ring around the parasite. The microgametocyte takes a lighter stain and the nucleus occupies half the parasite. The color with Giemsa appears light pink toward the edge. The parasite is completely enclosed in a prominent circle of eosinophilic stippling.
Molecular techniques for the differentiation of P. ovale from other species of human malaria parasites have been developed using PCR. Snounou et al. (89) were the first to apply the two-step nested PCR technique to the separation of all four human infecting species using the P. ovale primers rOVA 1 (ATC TCT TTT GCT ATC TTT TTT TAG TAT TGG AGA) and rOVA 2 (GGA AAA GGA CAC ATT AAT TGT ATC CTA GTG). In the first step (PCR1), extracted DNA is amplified using genus-specific primers; in the second step (PCR2), the PCR1 amplification product is further amplified using species-specific primers. Then, each PRC2-amplified DNA product is separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV illumination. The migration position on the gel identifies the species of Plasmodium present. The P. ovale primers described on the Centers for Disease Control and Prevention DPDx Website are those presented by Snounou et al.
Oliveira et al. (79) reported a procedure where the target region of the 18S rRNA gene is amplified by PCR using an 18S rRNA, genus-specific, biotinylated (5') and an unlabeled primer (3') pair. The detection probes were digoxigenin-labeled DNA oligonucleotides derived from species-specific rRNA sequences. The amplified fragment complex is allowed to hybridize with the species-specific, digoxigenin-labeled oligonucleotide probes. The oligo/DNA complex is allowed to bind onto streptavidin-peroxidase substrate. The two different pairs of primers were used to detect P. ovale were DIG 11 (5' AAT AAG AAC ACA TTT TGC A) and DIG 12 (3' CAG ATA CGT TGT ATT GTC) and DIG 13 (5' AAT AGC AAA AGA GAT TTT) and DIG 14 (3' CAT CTT ATA GCA AAA GTA).
The preservation of viable malaria parasites was a major breakthrough in the study of these organisms. In 1955, Jeffery and Rendtorff (56) reported the frozen preservation of both blood and sporozoite stages of P. ovale. Blood stages were stored for up to 234 days at a temperature of 70°C. Sporozoites were also readily preserved following dissection into human plasma and subsequent storage at –70°C. Suspensions of sporozoites were quickly thawed and then injected intravenously into recipient volunteers. Prepatent periods were similar to those of patients receiving infection via mosquito bite. In a subsequent report, (54) frozen preservation of the Donaldson strain of P. ovale was reported for periods of 399 and 997 days. Most infections with P. ovale in chimpanzees have been induced by the injection of infected erythrocytes that has been stored frozen over liquid nitrogen, often for many years (25, 76). Parasites are usually stored in Glycerolyte and are expected to be viable for decades when held at extremely low temperatures. Thick and thin blood films for immunofluorescence studies and teaching can be stored unfixed and frozen for extended periods. Frozen blood is unsuitable for preparation of blood films.