Abstract
BackgroundThe need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations.MethodsDNA was extracted from two RDT devices (Paracheck-Pf® and SD Bioline Malaria Pf/Pan®), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman® 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance.ResultsThe P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples.ConclusionsThe results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.
Highlights
The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts
Comprehensive studies are needed to investigate whether wide-scale collection of rapid diagnostic tests (RDTs) can provide the basis for modern molecular surveillance of malaria, including both improved malaria case detection and anti-malarial drug resistance genotyping
In vitro cultured parasites - sensitivity of RDT-DNA extraction methods The P. falciparum DNA detection limit varied with RDT device and extraction method (Table 1)
Summary
The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Thereafter, two additional methods of DNA extraction from RDTs have been published [3,4] suggesting that RDTs are a reliable source for parasite DNA preservation This provides an opportunity for improved molecular surveillance and RDT quality control. Comprehensive studies are needed to investigate whether wide-scale collection of RDTs can provide the basis for modern molecular surveillance of malaria, including both improved malaria case detection and anti-malarial drug resistance genotyping
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