Abstract
BackgroundThis study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR.MethodsFirst, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples.ResultsBest results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests.ConclusionsRDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT fragment sampling for a wide range of RDT brands in combination with a simple and low cost extraction method, allowing RDT quality control.
Highlights
This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR
Rapid diagnostic tests (RDTs) are frequently used as an adjunct to microscopy in the diagnosis of malaria [1] and even as a point-of-care diagnostic tool [2]
Antigen detection was performed by two RDTs: 1) the SD-FK60 Malaria Ag Pf/pan test (Standard Diagnostics, Hagal-Dong, Korea, further referred to as SDFK60) detecting P. falciparum (Pf) histidine-rich protein-2 (HRP-2) and pan-species parasite lactate dehydrogenase, and, 2) the OptiMAL® pLDH (Pan, Pf) (Diamed AG, Cressier, Switzerland, further referred to as OptiMAL) targeting Pf-specific pLDH and pan-species specific pLDH
Summary
This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR. World Health Organization (WHO) recommends the use of RDTs as part of parasite-based diagnosis and supports the broad implementation of RDTs for malaria diagnosis in areas where malaria is Recently, a species-specific Plasmodium real-time PCR was successfully applied on stained thick blood films as the source of DNA. Such PCR on slides can have applications in clinical and research settings in case whole blood samples are not available [9,10]. The use of stored RDTs as source of DNA for PCR amplification might obviate the need for collection of whole blood or filter-based blood samples
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