The development of methods to amplify specific segments of DNA, of which the earliest and most commonly used is the polymerase chain reaction (PCR), has made it possible for the first time to conduct detailed molecular investigations of malaria parasites from a few microlitres of infected blood. Indeed, the specificity and sensitivity of PCR are such that DNA fragments can be routinely amplified from a blood aliquot that contains a few parasites only. Finger-prick samples, the simplest and least invasive sampling procedure, which hitherto served almost exclusively to generate blood smears for microscopic examination, thus became the collection method of choice for molecular studies of Plasmodium parasites based on PCR amplification. The demonstration that successful PCR analyses can be performed from DNA extracted from few drops of bloods dried on a filter paper that have been kept and shipped at room temperature
 revolutionized molecular epidemiological studies of malaria. Typically 20 μl to 50 μl of blood are obtained and blotted directly on a filter paper that is then left to dry before being individually sealed with an inert desiccant in a plastic bag. Given the ease of collection and that no cold chain is needed, this procedure has been almost universally adopted for field-based investigations. Moreover, dried blood spots can be stored in a fridge or even at room temperature until DNA extraction. The most common uses for this DNA are: parasite detection and identification, investigation of parasite population structure based on the genetic diversity of selected loci, analysis of mutations associated with drug resistance, and finally Plasmodium genotyping to correct outcome of in vivo drug trials in endemic settings.
It has now become customary to include dried blood spots collection in many trials and field surveys, and after the primary aims of the study have been achieved, duplicate spots are often retained as archival material, usually stored at room temperature. These dried blood spot collections afford an opportunity to conduct retrospective molecular epidemiological investigations on malaria parasites. There is evidence to suggest that the suitability of DNA obtained from dried blood spots may be affected not only by the extraction method, but also by the type of filter paper used as well as the storage temperature and duration
[2–4]. In these few studies that have examined the effect of such long-term storage on the efficacy of DNA amplification, the results were somewhat contradictory. A loss of PCR sensitivity was observed in two studies
[2, 4], while some improvement in this sensitivity was noted in the third
Over the years, researchers at the Shoklo Malaria Research Unit (SMRU) amassed a substantial collection of dried blood spots collected from patients recruited to a wide spectrum of clinical, chemotherapeutic and epidemiological investigations on malaria in populations in and around Mae Sot, a large town on the Thai-Myanmar border. The epidemiology of malaria parasites has changed considerably during this period. For example, Plasmodium falciparum is no longer the dominant species, and the pattern of resistance to various anti-malarial drugs has shifted such that P. falciparum parasites are now resistant even to artemisinin derivatives therapy (ACT)
 and resistance to chloroquine is emerging in Plasmodium vivax. Concurrently, technical advances have led to a massive increase in sequence data from malaria parasites. The complete genomes of P. falciparum and P. vivax are now available
[7, 8], as are extensive sequence data from an increasing number of different strains from these two species
[9, 10]. This has opened the way to assess the significance of genetic polymorphisms predicted or found associated to drug resistance, as was the case for resistance to the artemisinins
, clinical evolution or the acquisition of immunity. In this context, the dried blood spots collected over the years represent a precious parasite DNA repository
The dried blood spots have been stored at ambient temperature under normal atmospheric conditions. Thus, in order to ensure that they could serve as a reliable source of Plasmodium DNA for further investigations, it was necessary to establish whether storage has led to a loss in the ability to amplify specific DNA fragments by PCR. To address this issue, subsets of dried blood spots collected over 12 years starting from 1998 from patients diagnosed with P. falciparum that were recruited to various randomized trials were tested.