Background
The availability of sensitive diagnostic tools for malaria is critical to ensure appropriate treatment for patients and to preserve the lifespan of effective anti-malarials. In the field, the most common tools for malaria diagnosis are microscopy and rapid detection tests (RDTs), which are performed directly from the blood sample. Molecular methods that amplify and detect
Plasmodium DNA using specific reagents and platforms, such as real-time PCR, provide far greater sensitivity, but are not yet usable at the point-of-care. However, these methods have important applications in clinical research studies that involve the analysis of blood samples collected in the field, including genotyping parasite populations and monitoring drug resistance, genetic characterization of vaccine candidates, anti-malarial efficacy trials and surveillance programs [
1‐
3].
The performance of molecular tests largely depends on the quality of the parasite DNA. Highly purified DNA requires laborious sample processing and costly reagents, kits or equipment, whereas cruder extraction methods often produce DNA that is insufficiently pure for downstream testing. The presence of PCR inhibitors from the blood, such as haemoglobin, reduces the efficiency of the molecular reaction and compromises sensitivity [
4,
5]. However, the discovery of DNA polymerases that are resistant to PCR inhibition enables DNA to be amplified from blood without prior extraction. For malaria, this was recently demonstrated using the Phusion™ enzyme which amplifies DNA by nested PCR directly from dried blood spots on filter papers [
6].
One of the major advances in molecular diagnostics is the integration of fluorescence-based detection of DNA in real-time PCR. This poses a new challenge to direct PCR from blood as fluorophores are quenched in the presence of haemoglobin. Amplification can be achieved, but the product is not detected. One approach to overcome the quenching effect uses inhibitor-resistant
Taq mutants in combination with an enhancer cocktail within the PCR master mix for optimal amplification and fluorescence detection [
7,
8]. With these reagents, real-time PCR can be performed even with 25% blood volume in the PCR reaction [
8]. The usefulness of this method was evaluated for the direct detection of
Plasmodium DNA by real-time PCR from raw patient samples and from dried blood spots collected in the field.
Discussion
This report describes a methodology for DNA amplification directly from blood that is compatible with fluorescence detection by real-time PCR. Although this method cannot be used directly in the field, it facilitates the analysis of blood samples collected in field studies. Clinical trials on antimalarial or vaccine efficacy and prevalence or surveillance studies are just a few examples of applications of this test for high throughput analysis of blood samples. Either blood spots or whole blood samples can be transported to the lab and immediately tested by real-time PCR without the need for DNA extraction.
This method is particularly suited to studies seeking a rapid confirmation of malaria. The sensitivity is similar to real-time PCR performed on purified DNA and specificity is achieved through primer design to reduce non-specific amplification. At the Alberta ProvLab, malaria diagnosis is confirmed by a TaqMan real-time PCR assay that uses a previously published primer set for the
Plasmodium screening reaction [
16]. The primers from this assay were initially tested in the assay described here for real-time PCR from blood. Although amplification of
Plasmodium DNA was achieved with these primers with good sensitivity, sporadic non-specific amplification was observed in the negative controls. Use of the primers recently published by Kamau et al. [
13] reduced the formation of non-specific products. For some of the species identification reactions, improvements to the primer design may further reduce non-specific products that are detected as very late amplification curves. Furthermore, primers can be designed to produce amplicons with different melting curves for each species which would enable the species to be identified in a single multiplex reaction and further reduce the cost per test.
In general, the background fluorescence in this assay is higher than in other real-time PCR assays; the high concentration of SYBR® Green required to overcome the quenching effect from the blood results in higher background fluorescence and the threshold was set at 20,000 or greater for these assays. With blood spots, fluorescence from the filter paper itself resulted in even higher background. Therefore, preliminary testing with blank filter papers and spots of uninfected blood is highly recommended to define the Tm and expected melt curve for a positive sample relative to background. Once this is established, the interpretation of results is straight-forward. Another important consideration is that the real-time PCR must be performed in tubes and not capillaries. Preliminary testing in capillaries on the LightCycler instrument from whole blood samples failed to generate amplification curves. It is hypothesized that during the course of the PCR, dried blood accumulates along the walls of the capillaries and blocks the emission of fluorescence.
In addition to the detection of
Plasmodium DNA in blood, the reagents for direct PCR can also support the detection of other blood-borne pathogens. These reagents have previously been used to detect herpes simplex virus 2 and other viruses spiked into blood [
8]. In malaria-endemic countries, the diagnosis of non-malarial causes of fever is critical to prevent morbidity and mortality, particularly from bacterial infections. A recent meta-analysis identified
Streptococcus pneumoniae,
Salmonella enterica,
Staphylococcus aureus,
Escherichia coli and
Haemophilus influenzae as the major causes of bloodstream infections in African children [
17]. The PCR test described here could be developed to detect these other organisms directly from blood samples.
In addition to the practical use of this assay for the qualitative analysis of field samples, the ability to perform real-time PCR for malaria directly from blood can be exploited in new technologies to bring molecular diagnostics for malaria to the point-of-care [
18]. A number of technologies have been reported but they generally require an upstream module for DNA extraction [
3,
19,
20]. Using the methodology described, this step can be omitted and samples can be immediately processed for diagnostic testing.
Conclusions
The methodology described here supports the amplification of gDNA by fluorescence-based real-time PCR directly from blood samples infected with malaria. The advantage of this assay is the elimination of the DNA extraction step, thereby facilitating high-throughput analysis of samples collected in the field using state-of-the-art tools for the detection and characterization of Plasmodium infections.
Acknowledgements
We thank Sandra Shokoples for laboratory assistance and Severiano Velazquez for field collection assistance. We are grateful to Dr. John Crabtree and Dr. Linda Pilarski for comments on the manuscript. The following reagents were obtained through the MR4: genomic DNA from Plasmodium knowlesi H strain and Plasmodium falciparum 3D7 malaria parasites contributed by D. J. Carucci. This work was supported by an Interdisciplinary Team Grant from the Alberta Heritage Foundation for Medical Research, Colciencias (contract RC 238-2007) and the Universidad de Antioquia.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
BJT and KAM performed the real-time PCR testing of blood samples and contributed equally to this work. AM provided dried blood spots from samples collected in the field and EA and OMA tested these samples by nested PCR. SKY and BJT designed the experiments and SKY wrote the manuscript. All authors read and approved the final manuscript.