This is to date the most comprehensive comparative study of DNA extraction efficiency from malaria RDTs and assessment of the field applicability of RDT-DNA extraction for molecular surveillance, including detection of infections and key genetic markers associated with anti-malarial drug resistance.
DNA extraction efficiency from in vitro cultured P. falciparum varied with RDT device and extraction method. The same level of parasite detection as seen in previous publications was not achieved in this study [3, 4]. Different designs of RDT devices affected the DNA extraction efficiency. In particular, DNA recovery from Paracheck-Pf was unsuccessful when employing the simple elution method, supporting as previously reported that plastic seals covering the nitrocellulose strip hamper DNA recovery .
The cost, time, final template volume and the purpose for DNA extraction should be considered when choosing extraction method. Although simple elution is the cheapest and fastest alternative, it is a crude method of DNA extraction and its use may be limited by RDT design and choice of PCR . Chelex-100 is relatively inexpensive. The higher sensitivity observed with Chelex-100 extraction indicates that this method is particularly suitable for low density parasitaemia in low endemic settings. However, the Chelex-100 method is moderately labour-intensive and the DNA may be of lower quality than DNA extracted with commercially available column-based extraction kits . Another concern is the storage capacity of Chelex-100 extracted DNA, which is thought to be more susceptible to DNA degradation during sample freeze-thawing . Conversely, ABI extraction is a high throughput method providing high quality DNA, but at a substantial cost and requiring specialised equipment. This method had a higher P. falciparum detection level when compared with Chelex-100, perhaps explained by loss of DNA on the column. However, this did not appear to have influenced the field sample results (see below). Thus, ABI extraction could be suitable for analyses of RDT positive, symptomatic malaria patients enrolled in clinical trials. The final DNA containing volume is also important to take into consideration, as the concentration of the DNA will affect the parasite detection limits.
In the field analysis, RDTs provided DNA of equal quality as filter papers, suggesting that RDTs are a valuable alternative to filter paper for DNA storage in the field. High PCR success rates were obtained from DNA extracted from RDTs, for key loci in pfcrt and pfmdr1 associated with anti-malarial drug resistance. SNP and haplotype prevalences were analysed and discussed elsewhere . Wide scale collection of used RDTs is currently being implemented as an integral part of molecular surveillance of malaria in Zanzibar.
Increased deployment of RDTs in health care facilities and cross-sectional surveys facilitates passive and active collection of biological material for molecular surveillance. The advantages of using RDTs for DNA storage include reducing invasive procedures in the field. RDTs require just one finger prick for both malaria case detection and preservation of biological material. DNA storage on filter papers, on the other hand, requires an initial finger prick for malaria case detection by RDT or microscopy, followed by a second finger prick, for individuals with a positive diagnosis for collection of blood on filter paper. Multiple blood sampling can especially be problematic in small children and may increase the risk of mixing/miss-labelling of samples during collection. RDTs are also easily stored and have either a plastic or cardboard case that protects against cross-contamination. A disadvantage of RDT-DNA extraction is the limited amount of biological material (5–15 μL blood). This makes RDT-DNA extraction a “one shot operation” with no possibilities for re-extraction, unlike filter paper sampling where a larger amount of blood is usually collected (50–100 μL).