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Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

Ulrika Morris1*, Berit Aydin-Schmidt1, Delér Shakely12, Andreas Mårtensson13, Louise Jörnhagen1, Abdullah S Ali4, Mwinyi I Msellem4, Max Petzold5, José P Gil678, Pedro E Ferreira19 and Anders Björkman1

Author Affiliations

1 Malaria Research, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden

2 Department of Medicine, Kungälv Hospital, Kungälv, Sweden

3 Department of Public Health Sciences, Division of Global Health (IHCAR), Karolinska Institutet, Stockholm, Sweden

4 Zanzibar Malaria Control Programme (ZMCP), Ministry of Health, Zanzibar, Tanzania

5 Centre for Applied Biostatistics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

6 Drug Resistance and Pharmacogenetics Group, Institute of Biotechnology and Bioengineering, Centre of Molecular and Structural Biomedicine, University of Algarve, Faro, Portugal

7 Laboratory of Molecular Anthropology and Health, Department of Anthropology, Binghamton University, Binghamton, NY, USA

8 Department of Physiology and Pharmacology, Drug Resistance Unit, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden

9 Department of Protozoology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan

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Malaria Journal 2013, 12:106  doi:10.1186/1475-2875-12-106

Published: 19 March 2013

Abstract

Background

The 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.

Methods

DNA 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.

Results

The 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.

Conclusions

The 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.

Keywords:
Plasmodium falciparum; Malaria; Rapid diagnostic test; DNA extraction; Molecular surveillance