A novel method for extracting nucleic acids from dried blood spots for ultrasensitive detection of low-density Plasmodium falciparum and Plasmodium vivax infections

Abstract

Background: Greater Mekong Subregion countries are committed to eliminating Plasmodium falciparum malaria by 2025. Current elimination interventions target infections at parasite densities that can be detected by standard microscopy or rapid diagnostic tests (RDTs). More sensitive detection methods have been developed to detect lower density "asymptomatic" infections that may represent an important transmission reservoir. These ultrasensitive polymerase chain reaction (usPCR) tests have been used to identify target populations for mass drug administration (MDA). To date, malaria usPCR tests have used either venous or capillary blood sampling, which entails complex sample collection, processing and shipping requirements. An ultrasensitive method performed on standard dried blood spots (DBS) would greatly facilitate the molecular surveillance studies needed for targeting elimination interventions.

Methods: A highly sensitive method for detecting Plasmodium falciparum and P. vivax 18S ribosomal RNA from DBS was developed by empirically optimizing nucleic acid extraction conditions. The limit of detection (LoD) was determined using spiked DBS samples that were dried and stored under simulated field conditions. Further, to assess its utility for routine molecular surveillance, two cross-sectional surveys were performed in Myanmar during the wet and dry seasons.

Results: The lower LoD of the DBS-based ultrasensitive assay was 20 parasites/mL for DBS collected on Whatman 3MM filter paper and 23 parasites/mL for Whatman 903 Protein Saver cards-equivalent to 1 parasite per 50 µL DBS. This is about 5000-fold more sensitive than standard RDTs and similar to the LoD of ≤16-22 parasites/mL reported for other ultrasensitive methods based on whole blood. In two cross-sectional surveys in Myanmar, nearly identical prevalence estimates were obtained from contemporaneous DBS samples and capillary blood samples collected during the wet and dry season.

Conclusions: The DBS-based ultrasensitive method described in this study shows equal sensitivity as previously described methods based on whole blood, both in its limit of detection and prevalence estimates in two field surveys. The reduced cost and complexity of this method will allow for the scale-up of surveillance studies to target MDA and other malaria elimination interventions, and help lead to a better understanding of the epidemiology of low-density malaria infections.

Keywords: Asymptomatic infection; DBS; Diagnostics; Dried blood spot; Limits of detection; Low transmission; Malaria; Malaria elimination; Molecular surveillance; Myanmar; Plasmodium falciparum; Plasmodium vivax; Southeast Asia; Ultrasensitive PCR.

Fig. 1

Schematic representation of the new extraction method. A detailed protocol can be found in Additional file 1

Fig. 2

Map indicating sites in Myanmar where samples were collected during the wet and dry season

Fig. 3

Sample collection and contamination minimization strategies. a Whatman 3MM filter paper is pre-cut to yield four 2 cm × 0.5 cm strips, each which corresponds to a 50 µL blood spot. Four horizontal cuts across a strip yield small enough pieces to fit into a standard 96 well plate (which has already been performed for the missing strip on the right), thereby reducing cutting time. b Plastic plate covers used to prevent cross-contamination. On the left is a typical plastic plate cover used during extraction and PCR setup which allows multichannel pipetting of an entire column (eight wells) but covers the remainder of the 96 well plate. The cover on the right is for a single well of a 96 well plate and is used during cutting to isolate the well receiving the cut DBS

Fig. 4

Controlling for contamination and false positives. a Wiping scissors and forceps three times with a kimwipe sprayed with 70% ethanol is sufficient to prevent contamination when cutting from a high to a zero parasitemic sample. b The amplification curves for all of the 0 parasites/mL samples are shown indicating lack of amplification. c Representative plates from a field survey from Myanmar showing that even in high malaria burden areas, negative control samples consisting of blank filter papers (indicated by asterisk) remain negative