Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots

Abstract

BackgroundBlood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known.MethodsDNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number.ResultsWhen a 1.6 kb fragment of the parasites’ small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted.ConclusionsThe average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.

The Effect of Storage and Extraction Methods on Amplification of Plasmodium falciparum DNA from Dried Blood Spots

“…Our findings are in agreement with two prior studies that showed decreased PCR sensitivity for microscopically detectable infections after 4 and 7 years of storage. 12,13 Thus, studies attempting to determine the prevalence of parasitemia by PCR, especially from subjects who are likely to have low parasite densities (e.g., asymptomatic subjects or those who are negative by microscopy or rapid diagnostic test), should be interpreted cautiously if DBS samples have been stored at ambient temperature for more than 2 years. On the basis of our results, if DBS samples are not analyzed promptly, they should be stored at −20 C or DNA should be extracted before storage.…”

Characterising temporal trends in asymptomatic Plasmodium infections and transporter polymorphisms during transition from high to low transmission in Zanzibar, 2005–2013

“…The supernatant containing genomic DNA was stored at À20°C until use; PCR screening was conducted within 3 months of DNA extraction. Older samples collected in 2005, which were positive in the initial real-time PCR screening, were re-extracted with the QIAmp DNA mini kit (Qiagen Gmbh, Germany) in order to improve the quality of the DNA (Hwang et al, 2012). DNA was extracted from three 3 mm filter paper discs according to the protocol ''DNA purification from dried blood spots'' (Qiagen, 2012).…”

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

“…However, and importantly, comparative studies on the efficiency of the published DNA extraction methods from RDTs are lacking. This is critical since there is evidence that the efficiency of DNA extraction may be highly influenced by choice of extraction matrix and method [3-6]. Furthermore, 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.…”