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Comparative Study
. 2017 Aug;97(2):533-543.
doi: 10.4269/ajtmh.17-0039. Epub 2017 Jul 19.

A Direct From Blood Reverse Transcriptase Polymerase Chain Reaction Assay for Monitoring Falciparum Malaria Parasite Transmission in Elimination Settings

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Free PMC article
Comparative Study

A Direct From Blood Reverse Transcriptase Polymerase Chain Reaction Assay for Monitoring Falciparum Malaria Parasite Transmission in Elimination Settings

Brian J Taylor et al. Am J Trop Med Hyg. .
Free PMC article

Abstract

We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for Plasmodium falciparum malaria parasites that amplifies RNA targets directly from blood. We developed the assay to identify gametocyte-specific transcripts in parasites from patient blood samples, as a means of monitoring malaria parasite transmission in field settings. To perform the test, blood is added directly to a master mix in PCR tubes and analyzed by real-time PCR. The limit of detection of the assay on both conventional and portable real-time PCR instruments was 100 parasites/mL for 18S rRNA, and 1,000 parasites/mL for asexual (PFE0065W) and gametocyte (PF14_0367, PFGEXP5) mRNA targets. The usefulness of this assay in field studies was explored in samples from individuals living in a high-transmission region in Cameroon. The sensitivity and specificity of the assay compared with a standard two-step RT-PCR was 100% for 18S rRNA on both conventional and portable instruments. For PF14_0367, the sensitivity and specificity were 85.7% and 70.0%, respectively, on the conventional instrument and 78.6% and 90%, respectively, on the portable instrument. The concordance for assays run on the two instruments was 100% for 18S rRNA, and 79.2% for PF14_0367, with most discrepancies resulting from samples with low transcript levels. The results show asexual and sexual stage RNA targets can be detected directly from blood samples in a simple one-step test on a field-friendly instrument. This assay may be useful for monitoring malaria parasite transmission potential in elimination settings, where sensitive diagnostics are needed to evaluate the progress of malaria eradication initiatives.

Conflict of interest statement

Conflict of interest: Stephanie K. Yanow is a member of the Scientific Advisor Board of Aquila Diagnostic Systems, Inc.

Figures

Figure 1.
Figure 1.
Plasmodium falciparum 18S rRNA detection in blood by direct reverse transcriptase polymerase chain reaction (RT-PCR). (A) Real-time amplification and (B) melting curves for 18S rRNA direct RT-PCR. Total nucleic acid was first purified from serial dilutions of P. falciparum 3D7 ring-stage parasites in uninfected blood. This purified nucleic acid was added back to blood in the direct RT-PCR reaction. (C) Amplification and (D) melting curves for 18S rRNA using the intact unpurified parasite serial dilutions in blood added directly to the reaction. The limit of detection for 18S rRNA from purified parasite nucleic acid is 10 parasites/mL and from unpurified parasite serial dilutions is 100 parasites/mL. These reactions were performed on a conventional real-time PCR instrument (Bio-Rad CFX Connect) with a threshold setting of 50 relative fluorescence units (horizontal line). This figure appears in color at www.ajtmh.org.
Figure 2.
Figure 2.
Detection of Plasmodium falciparum PFE0065w (SBP1) mRNA from asexual stages in blood. (A) Real-time amplification and (B) melting curves for PFE0065w mRNA direct reverse transcriptase polymerase chain reaction (RT-PCR) using P. falciparum 3D7 ring-stage serial dilutions in blood. The limit of detection for PFE0065w mRNA is 1,000 parasites/mL. This reaction was performed in the conventional real-time PCR instrument.
Figure 3.
Figure 3.
Detection of Plasmodium falciparum gametocyte mRNA targets in blood. (A) Real-time amplification and (B) melting curves for PF14_0367 mRNA direct reverse transcriptase polymerase chain reaction (RT-PCR). (C) Real-time amplification and (D) melting curves for PFGEXP5 mRNA direct RT-PCR. Both mRNA targets were detected in P. falciparum NF54 gametocyte serial dilutions on the conventional instrument at a limit of detection of 1,000 parasites/mL. This figure appears in color at www.ajtmh.org.
Figure 4.
Figure 4.
Detection of Plasmodium falciparum PF14_0367 mRNA and 18S rRNA in sexual- and asexual-stage parasites by direct RT-PCR. Plasmodium falciparum strain NF54 stage V gametocytes and asexual ring stages purified 10 hours after merozoite invasion at 1 × 106 parasites/mL blood. Arrows indicate the amplification curves for 18S rRNA in gametocytes and ring stages (0.8 cycle shift), and Pf14_0367 (8.3 cycle shift).
Figure 5.
Figure 5.
Detection of Plasmodium falciparum 18S rRNA and PF14_0367 mRNA in a portable real-time PCR instrument by direct RT-PCR. Plasmodium falciparum RNA targets were tested in a 16-well portable real-time PCR instrument. Amplification (A, C, and E) and melt curve (B, D, and F) data from the instrument are shown. For the18S rRNA assay, standard (1×) concentration of enzyme was used, and produced a LOD of 100 parasites/mL (A and B). For the PF14_0367 assay, mRNA was detected in 3D7 gametocytes at a LOD of 10,000 parasites/mL with 1× concentration of enzyme (C and D) or 1,000 parasites/mL with 5× concentration of enzyme (E and F). Individual Cq values obtained directly from the instrument are listed in Supplemental Table 2. This figure appears in color at www.ajtmh.org.

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