Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum

Abstract

Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 microl of venous blood to achieve this sensitivity, compared to 50 microl of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.

FIG. 1.

(a) Mean number of P. falciparum ring stage parasites (circles) and gametocytes (squares) in a 10-fold dilution series quantified by real-time 18S rRNA QT-NASBA. Error bars, standard deviations. The log number of parasites per sample as counted by microscopy shows a highly significant correlation with the time to positivity (TTP) as calculated by real-time QT-NASBA (for ring stage parasites, R² = 0.942 and P < 0.01; for gametocytes, R² = 0.966 and P < 0.01). (b) Mean number of P. falciparum ring stage parasites in a 10-fold dilution series quantified by 18S real-time QT-PCR. The mean is calculated for 7 observations with 10 parasites/ml, 12 observations with 10² parasites/ml, and 18 observations with 10³ to 10⁶ parasites/ml. Error bars, standard deviations. The log number of parasites per sample as counted by microscopy shows a highly significant correlation with the threshold cycle as calculated by real-time QT-PCR (R² = 0.985; P < 0.01).

FIG. 2.

Quantification of parasites in blood samples taken from a volunteer during a human experimental P. falciparum infection. Mean results from duplicate analyses are shown for real-time QT-NASBA (open circles) and real-time QT-PCR (solid squares). The results of quantification by the two techniques were significantly correlated (Spearman's ρ = 0.930; P < 0.01). Drug treatment was given at day 8.3 after infection, when parasites were detected by microscopy.