Quantitation of Liver-Stage Parasites by Automated TaqMan® Real-Time PCR

Abstract

The liver stages of the malaria parasite have long been a difficult part of the life cycle to study. Very little is known about the localization of the parasite within the liver and no method for the estimation of liver parasite burden is in common use. Differences in liver parasite burden would provide a good measure of preerythrocytic malaria vaccine efficacy, which currently is measured indirectly by the counting of blood-stage parasites that develop following a sporozoite challenge. However, the measurement of bloodstage parasitemia is a slow, painstaking process requiring a high level of technical ability. In addition, a biological amplification of parasites occurs in hepatocytes, with one infected hepatocyte yielding approx 4000 -0,000 merozoites, suggesting that counting blood-stage parasitemia may not be an accurate way of quantifying preerythrocytic stage vaccine efficacy. A direct method would, therefore, be preferable. Several methods have been described to attempt to examine liver-stage parasitemia directly, including the use of radioactive oligonucleotides to probe RNA blots (1), high performance liquid chromatography (HPLC) (2), or competitive polymerase chain reaction (PCR) to quantitate PCR products generated to specific parasite targets (3). However, these methods are cumbersome and not conducive to high-throughput screening procedures. The TaqManR realtime fluorescent PCR method described here is a fast and reproducible method for analyzing liver-stage parasitemia in rodent malaria models. The method has previously been used to investigate pre-erythrocytic malaria vaccine efficacy (4).