Nearly all malaria-associated mortality in children is due to infection with Plasmodium falciparum
, which causes over 300 million clinical infections and a million deaths per year in African children under five years of age [1
]. Genome sequencing of multiple parasite isolates [2
] indicates that the parasite population is highly diverse. Genetic diversity in P. falciparum
is manifested in the form of single nucleotide polymorphim (SNPs), microsatellite repeats, insertions, deletions and a range of gene duplication events. Much of this diversity segregates independently. Analysis of the progeny from a genetic cross suggests that the parasite genome is approximately 50-fold more 'recombinogenic' than the human genome [2
]. This genetic diversity underlies the ability of the parasite to escape both immune clearance and drug treatment.
Parasite genetic variation can be exploited epidemiologically to provide a means of uniquely identifying parasites that infect individuals and to follow these parasites through the course of infection as drug or other interventions are applied. For example, these methods are useful for identifying parasites in an individual prior to treatment to later determine if post-treatment parasitaemia are the result of failure to clear the original parasites by the drug(s) (recrudescence) or of re-infection by another parasite form. These methods are critical for determining the efficacy of drugs in the field.
The first powerful and easily deployable tool for assessing the identity (i.e., genotype) of a given parasite isolated from a patient was published in 1994 by Snounou using three polymorphic loci (merozoite surface protein (msp
-2, and glutamine rich protein, glurp
) in a nested polymerase chain reaction (PCR) to identify length polymorphisms within six potential alleles (K1, MAD20, and RO33 for msp-1
; IC and FC27 for msp-2
; and glurp
]. The technique requires gel electrophoresis, fluorescent primers in conjunction with capillary electrophoresis to resolve the length of the individual allele polymorphisms, or a Luminex fluorescent microsphere assay [4
]. The practical use of this genotyping system – whole or in part – was reviewed by Collins et al
for 91 of 384 studies between 1995 and 2005, focused on antimalarial clinical trials [5
]. They conclude from this meta-analysis that the great deal of variation found in the use and interpretation of this system was statistically related in multivariate analysis to the polyclonality of infections, treatment employed, geographic location, and duration of follow-up [5
]. Pyrosequencing has also been used to evaluate short stretches of DNA sequence from patient samples surrounding a single locus to quantify alleles and haplotypes of MSP-1 in a population [6
]. A modification of the standard MSP/GLURP genotyping technique, the heteroduplex tracking assay, demonstrated an ability to track complex infections in an endemic area with high polyclonality (3.82 per patient). However, this assay currently depends on the use of radioactive tracers, making it difficult or impossible to employ in many field settings [7
]. Although others have offered refinements on these basic approaches or focused on microsatellite polymorphisms for determining polyclonality, or some combination thereof, the current approaches are either labour-intensive, difficult to carry out in a field setting, or require subjective interpretation [8
As part of an ongoing effort to map genomic diversity of P. falciparum
, genome sequencing has identified more than 112,000 SNPs from about 18 parasite genomes [10
]. Current technology has made genotyping of SNPs by real-time PCR using dual probes in an end-point detection assay a standard practice. We filtered the discovered SNPs to create a panel of genotyping assays capable of defining a "molecular bar code" or signature for a given malaria parasite. Ideal SNPs for such an assay panel segregate independently, are common (i.e. exhibit a high minor allele frequency (MAF)) and are broadly distributed across the genome. The assay method has to be easy to use, inexpensive, and applicable to a wide variety of both field and laboratory derived material. The TaqMan system was choosen as a genotyping methodology, with the eventual goal of developing these assays into a simple, end-point PCR process that could be performed in the field where a PCR machine and a plate reader were available.
This work describes the first P. falciparum molecular bar code composed of 24 SNPs that in combination create a unique fingerprint or signature for a parasite genome. This methodology can be applied to a variety of laboratory and field samples including direct culture-adapted material, genomic DNA, frozen blood from patients or filter-paper collected samples with over 99% success. This methodology is extremely sensitive, requiring only a small amount of input material. Human DNA within the sample does not interfere with the results. This molecular barcode is also capable of identifying mixtures of parasite genomes within samples that would otherwise be identified as single parasite infections by conventional msp-1 and msp-2 genotyping, and thus provides a robust, inexpensive, facile method for evaluating parasite genomes within patient samples.