METHODS: Thick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per microl blood, were examined.

RESULTS: The P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae.

METHOD: One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level.

RESULTS: Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments.

Methods: The clinical information of the patient was collected. Microscopy of blood smear was conducted after Giemsa staining. Genomic DNA was extracted from blood, and PCR was conducted to amplify rDNA. The PCR products were sequenced and analyzed with BLAST

Results: The patient returned from a one-week tour in a tropical rain forest in Malaysia. The first disease attack occurred in Guangzhou on Oct. 16, 2014, with fever, shivering and sweating. The patient was initially diagnosed as malaria and hospitalized on Oct. 26, 2014. Microscopic observation revealed typical forms of P. knowlesi in blood smear. The red blood cells became enlarged, with big trophozoites appearing as a ring with dual cores and dark brown malaria pigment. The trophozoites were slightly bigger and thicker than P. falciparum. The schizont had 6-8 merozoites, with obvious brown malaria pigment. PCR resulted in a specific band of 1 099 bp. BLAST analysis showed that the sequence of the PCR product was 99% homologous to P. knowlesi (acession No. AM910985.1, L07560.1 and AY580317.1). The patient was diagnosed as P. knowlesi infection, and was then given an 8-day treatment with chloroquine and primaquine, together with dihydroartemisinin piperaquine phosphate tablet. The patient was discharged after recovery on Oct. 28, 2014.