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.
CONCLUSION: Plasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.
METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.
RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.
CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.