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Protection against a homologous malaria challenge can be induced by the inoculation of intact sporozoites. (ClinicalTrials.gov number, NCT00442377.)

Abstract. A nested polymerase chain reaction (PCR) assay that uses Plasmodium genus-specific primers for the initial PCR (nest 1) amplification and either genus-or species-specific primers for the nest 2 amplifications was tested on laboratory and field samples. With in vitro cultured Plasmodium falciparum-infected blood samples, it was capable of detecting six parasites/l of blood using DNA prepared from 25-l blood spots on filter paper. The assay was evaluated on fingerprick blood samples collected on filter paper from 129 individuals living in a malaria-endemic area in Malaysia. Malaria prevalence by genus-specific nested PCR was 35.6% (46 of 129) compared with 28.7% (37 of 129) by microscopy. The nested PCR detected seven more malaria samples than microscopy in the first round of microscopic examination, malaria in three microscopically negative samples, six double infections identified as single infections by microscopy and one triple infection identified as a double infection by microscopy. The nested PCR assay described is a sensitive technique for collecting accurate malaria epidemiologic data. When coupled with simple blood spot sampling, it is particularly useful for screening communities in remote regions of the world.Microscopy is the method of choice for the diagnosis of malaria in endemic areas because it is an inexpensive and rapid method of detection. Correct identification of the four species of Plasmodium causing human malaria and the level of detection by microscopy depends on a number of factors, including the experience of the microscopist, proper staining of the slides, appropriate maintenance of microscopes, and the time spent examining each slide. At best, the sensitivity of detection by microscopy is approximately 10-30 parasites/l of blood. 1 However, this level of detection is normally not attained in malaria-endemic areas and particularly during epidemiologic studies when many samples need to be screened in a relatively short time. Thus, incorrect speciation is common and mixed infections and low levels of parasitemia may be missed.To overcome some of the limitations of microscopy for detection of malaria, polymerase chain reaction (PCR)-based assays have been developed for the detection and identification of malaria parasites. These methods have proved to be more specific and sensitive than conventional microscopy and some are reported to be able to detect as few as one parasite/l of blood.2-6 However, to attain such a high sensitivity, blood samples collected from individuals have to be processed immediately or stored at low temperatures and the steps involved in DNA template preparation were multistep, often requiring biohazardous chemicals. Since malaria remains a problem of underdeveloped and often remote areas of the world, it is important to couple PCRbased assays with simple sampling and DNA extraction methods to maximize the value of PCR assays. In a previous study, we coupled the nested PCR assay of Snounou and others 3 to blood collection on filter papers and a simple DNA e...

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp. Although a high degree of diversity characterized these isolates, the overall population structure of the parasites associated with patent malaria infections was observed to remain relatively stable over time. The highest degree of polymorphism was observed with msp2, and the mean number of lines per infection (multiplicity of infection) calculated with this marker was higher than that obtained using msp1 or glurp alone, or combined. Infections with > or = 2 parasite lines were seen in 76% of the samples, and were proportionally more numerous at the start and end of the rainy season. Two interesting exceptions to the random distribution were observed and involved 2 allelic variants which in one case were found dissociated (msp1 MAD20-family) and in the other were associated (msp2 FC27-family). The epidemiological significance of these types of data is discussed.