An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples

Abstract

BackgroundHighly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.MethodsA reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.ResultsLimits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80 % humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.ConclusionsThis ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1038-z) contains supplementary material, which is available to authorized users.

Plasmodium malariaePrevalence andcspGene Diversity, Kenya, 2014 and 2015

“…malariae infections in samples with exceptionally low levels of parasitemia. More accurate prevalence data may be obtained from ultrasensitive PCR that targets multicopy regions of the parasite genome ( 45 ) or reverse transcription PCR of parasite RNA extracted from whole blood ( 46 ). …”

The Controlled Human Malaria Infection Experience at the University of Maryland

“…40,41 More recent studies at UMB-CVD have used novel ultrasensitive reverse transcription quantitative PCR (usRT-PCR) techniques, which has a sensitivity of ∼16 parasites/mL ( Figure 1B). 42 PCR primers for usPCR amplified the Plasmodium 18S ribosomal RNA genes, as well as the ribosomal RNA gene transcripts. 42,43 Daily results were analyzed data with the Roche LightCycler® 96 software version 1.1 or the Light-Cycler®480 software 1.5.1.…”

Submicroscopic and Asymptomatic Plasmodium Parasitaemia Associated with Significant Risk of Anaemia in Papua, Indonesia

“…Lower limits of detection range from 10–20 parasites/μL in research settings to more than 100/μL in clinical practice [4]. Recent advances in molecular techniques have allowed the development of highly sensitive methods for detecting peripheral parasitaemia at submicroscopic levels (0.1–10 parasites/μL) [5–8]. The application of molecular approaches in surveillance surveys has resulted in an inevitable increase in the prevalence of detectable parasitaemia, often 2–10 fold higher than the prevalence obtained by conventional methods [9].…”