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Invasion of the mosquito midgut by the Plasmodium ookinete determines the success of transmission of malaria parasites from humans to mosquitoes and therefore, is a potential target for molecular intervention. Here, we show higher-resolution ultrastructural details of developing and mature P. gallinaceum ookinetes than previously available. Improved fixation and processing methods yielded substantially improved transmission electron micrographs of ookinetes, particularly with regard to visualization of subcellular secretory and other organelles. These new images provide new insights into the synthesis and function of vital invasive machinery focused on the following features: apical membrane protrusions presumptively used for attachment and protein secretion, dark spherical bodies at the apical end of the mature ookinete, and the presence of a dense array of micronemes apposed to microtubules at the apical end of the ookinete involved in constitutive secretion. This work advances understanding of the molecular and cellular details of the Plasmodium ookinete and provides the basis of future, more detailed mechanistic experimentation on the biology of the Plasmodium ookinete.

Pooling clinical specimens reduces the number of assays needed when screening for infectious diseases. Polymerase chain reaction (PCR)-based assays are the most sensitive tests to diagnose malaria, but its high cost limits its use. We adapted a pooling platform that could reduce the number of assays needed to detect malaria infection. To evaluate this platform, two sets of 100 serum samples, with 1% and 5% malaria prevalence, were tested. DNA, extracted from pooled samples, was amplified by malaria-specific PCR. Additional validation was performed by determining the level of PCR detection based on 1:10 and 1:100 dilution. The platform correctly detected all malaria samples in the two test matrices. The use of stored serum samples also has important implications for studies investigating malaria prevalence rates retrospectively. Field studies, using serum and whole blood specimens, are needed to validate this technique for the adaptation of these methods for clinical utility.

Polymerase chain reaction (PCR) detection of Plasmodium DNA is highly sensitive in diagnosing malaria. The specimen of choice for this assay has been whole blood samples from malaria patients. To retrospectively determine malaria infection rates in populations or cohorts for whom stored serum samples are available, we determined the ability of a nested PCR assay to detect Plasmodium DNA in stored serum samples. The PCR result was positive in 20 of 23 serum samples from patients with microscopy-confirmed malaria and negative in 8 of 8 healthy controls, resulting in a sensitivity of 87% and specificity of 100%. In all positive samples, species were correctly identified by PCR except for one case where a mixed infection was detected. The PCR is able to detect Plasmodium DNA in serum samples frozen up to 2.5 years and has the potential for the retrospective identification of malaria parasitemia in patient cohorts to determine potential interactions of malaria and other diseases such as human immunodeficiency virus/acquired immunodeficiency syndrome.