A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots

Abstract

Background: As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material.

Methods: The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared.

Results: Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P<0.001) and symptomatic (Pearson's r=0.70, P<0.0001) P. falciparum infections.

Conclusion: Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.

Figures

Figure 1

Comparison of quantitative real-time PCR standard curves. 10-fold plasmid or gDNA dilutions were plotted against Ct values generated from qPCR only and nested qPCR assays using (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods). Points represent the mean of technical duplicates and error bars (where visible) indicate the standard deviation. Dotted lines represent the technical limit of detection as defined as the lowest template concentration for which there is a linear relationship between Ct values and copy number/parasite density. For (C), Ct values generated by nested qPCR were plotted against the parasite densities estimated from copy number for P. falciparum 18S rRNA plasmid or corresponding to the dilutions from the clinical DBS standards. The best-fit regression line is shown as a black line.

Figure 2

Amplification curves for nested qPCR assay. Nested qPCR amplification curves using 10-fold serial dilutions of (A)P. falciparum 18S rRNA plasmid or (B) gDNA extracted from dried blood spots obtained from a subject with clinical malaria as template DNA (described in the Methods).

Figure 3

Correlation between parasite densities determined by light microscopy and calculated by nested qPCR. Calculated parasite densities from nested qPCR were plotted against parasite densities determined by light microscopy for symptomatic infections (red circles), asymptomatic infections with positive blood smears (black circles), and asymptomatic infections with negative blood smears (unfilled circles). Parasite densities calculated by nested PCR strongly correlated with both asymptomatic (Pearson’s r = 0.58, 95% CI [0.29 to 0.77], P < 0.001) and symptomatic (Pearson’s r 0.70, 95% CI [0.53 to 0.81], P < 0.0001) P. falciparum infections. The dashed line represents the limit of detection for blood-smear microscopy (~40 parasites/μl).