Characterization of a Real-Time PCR Assay for the Detection and Quantification of Plasmodium malariae Parasites: Implications for Malaria Diagnosis
Parsons, Emily L.
MetadataShow full item record
With nearly one million deaths each year, malaria is a major world health issue (Guerra, et al., 2008, Kappe et al., 2010). Malaria is caused by the mosquito-transmitted protozoan parasite of the genus Plasmodium, four species of which cause malaria in humans. Low parasite densities can cause morbidity, so malaria diagnoses need to be sensitive to these ranges. Diagnoses must also be species-specific, because drug therapies differ for each species. In an effort to create sensitive and specific diagnostic tools, quantitative real-time polymerase chain reaction (RT-PCR) assays have been developed to detect malaria parasites. The most commonly used RT-PCR assays detect Plasmodium 18S rRNA and are highly sensitive. However, false positives have been reported with 18S rRNA assays (Vo et al., 2007). A new set of RT-PCR assays, developed by Vo et al. (2007), are more specific but are not as sensitive as the 18S assays. We have modified the assay developed by Vo et al. (2007) for P. malariae, creating an RT-PCR assay that is both specific and sensitive. This assay amplifies the P. malariae circumsporozoite gene (CS), using a sequence-specific probe for detection. We have thoroughly characterized this assay and show that is 100 % specific and maintains high species-specificity even in the overwhelming presence of additional Plasmodium species. With limits of detection and limits of quantitation at 1.25 and 3 parasites/μl, respectively, this assay is more sensitive than the Vo et al. (2007) assay. We have demonstrated this assay to be precise, accurate, robust, and suitable to any system in which specific species detection is a high priority. This assay is especially applicable to research settings with high through-put, providing more accurate diagnoses and assisting the malaria eradication agenda.