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

Abstract

We describe a novel one-step reverse transcriptase real-time PCR (direct RT-PCR) for 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.

RETRACTED ARTICLE: Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

“…Treated subjects carrying gametocytes in their blood are potential reservoirs of the parasite [7]. The development of modern tools for the identification of these reservoirs is necessary to achieve the elimination of malaria [4,6,8].…”

Molecular detection and quantification of Plasmodium falciparum gametocytes carriage in used RDTs in malaria elimination settings in northern Senegal

“…According to the National Malaria Control Program, this district has for many years recorded the vast majority of reported cases in the region where the transmission of the disease is strong at the local level [3]. Future studies involving asymptomatic patients are needed to evaluate the sensitivity and accuracy of the method and to evaluate the potential benefits of molecular control of transmission [8]. Extension of this study to the 3 regions where the malaria elimination project is operational will provide reliable estimates to better guide malaria control strategies [1].…”

“…Extension of this study to the 3 regions where the malaria elimination project is operational will provide reliable estimates to better guide malaria control strategies [1]. The use of RDTs as a source of DNA for the detection of mature gametocytes by molecular techniques is relevant for epidemiological studies assessing the efficacy of candidate vaccines against malaria transmission [8,21] and it is, therefore, an important support for controlling the reintroduction of malaria [8,9].…”

“…Treated subjects carrying gametocytes in their blood are potential reservoirs of the parasite [7]. The development of modern tools for the identification of these reservoirs is necessary to achieve the elimination of malaria [4,6,8].…”

“…Since mosquito infection requires the presence of gametocytes in humans, knowledge of the epidemiology of gametocyte carriage is an essential parameter for assessing malaria transmission and predicting infectiousness in vectors [1,7,8]. Some molecular techniques are used today for the detection and quantification of gametocytes [9,10].…”