BACKGROUND
The complete sequence of the malaria parasite, Plasmodium falciparum was published in October 2002(1). This unprecedented view into the genetic details of the malaria parasite was a significant event in parasitic research. Researchers were now faced with the challenge of finding meaningful information within the approximately 23Mb of DNA sequence contained in the P. falciparum genome. Novel genetic analysis tools were necessary to extract, analyze and interpret the information contained in the P. falciparum genome in a cost-effective and high-throughput fashion. The sections below will introduce genetic analysis tools, such as DNA microarrays, and illustrate how these tools can be applied to three different phases of malaria research: discovery, diagnostics and genetic surveillance.
Genetic Analysis Tools Enabling the Malaria Postgenomic Era
Genome sequencing efforts stimulated the development of advanced tools for genetic analysis. Microarray technology enables researchers to examine and mine data on a genome-wide scale. Experiments that previously took months, even years, to complete can now be finished in a matter of days.
Images courtesy of Illumina, Inc. and Affymetrix, Inc.
Please see (2-4, 5A, 6-8) for additional details on microarray technology platforms.
The GeneChip® Plasmodium/Anopheles Genome Microarray, which was jointly designed by The Malaria Research Institute at Johns Hopkins Bloomberg School of Public Health and Affymetrix, Inc., can be used for a wide variety of experiments, including gene expression monitoring and SNP genotyping. This array includes probe sets to over 4,300 Plasmodium falciparum transcripts and approximately 14,900 Anopheles gambiae transcripts.
Anopheles gambiae and Plasmodium falciparum images courtesy of Anthony Cornel and bepast.org
RESEARCH APPLICATIONS IN THE POSTGENOMIC ERA
Microarrays, along with other genetic analysis tools, have enabled the malaria research community to study the Plasmodium falciparum parasite on a genome-wide level. These tools can be used for a number of applications, including basic discovery research of malaria disease pathogenesis, molecular diagnosis of drug resistance, and genetic epidemiology and surveillance studies. Specific examples of discovery, diagnostic and surveillance research will be presented below.
Images courtesy of Wellcome Trust Sanger Institute (microarray), Novartis Foundation for Sustainable Development (mother/child), and Global Atlas of Infectious Disease (map of chloroquine resistance in Rwanda).
DISCOVERY RESEARCH
Basic Research on Malaria Disease Pathogenesis
A number of researchers are conducting genome-wide expression analysis and genetic variation mapping studies of P. falciparum. Their findings have identified new drug and vaccine targets for malaria disease control and prevention.
Differential Expression Patterns Throughout the Malaria Life Cycle
The life cycle of the malaria parasite Plasmodium falciparum consists of multiple stages within both the Anopheles vector and the human host (see Parasite section). Using microarray technology, researchers determined the expression patterns of P. falciparum genes at different stages of its life cycle(9). The following stages of development were examined:
Figure 1 illustrates the findings of this gene expression study. 4557 genes were expressed in at least one stage of the life cycle.
Figure 1. Venn diagram of life cycle–regulated genes in P. falciparum. The solid circles denote genes that are considered expressed in the erythrocytic cell cycle (ET and ES), gametocytes (G), or sporozoites (S). Courtesy of Science Magazine(9).
Genes associated with protein synthesis were upregulated in the ring and trophozoite stages, while cell surface structure genes were upregulated in schizonts and sporozoites. Expression profiling studies of the asexual erythrocytic developmental cycle have shown a highly specialized gene regulation pathway, which is an excellent candidate for drug intervention(10). Scientists are now focusing on approximately 100 of the genes involved in the P. falciparum life cycle as targets for new antimalarial drugs and vaccines.
Mapping Genetic Variation in the Parasite (Plasmodium falciparum)
Recent studies have demonstrated a rich genetic diversity among P. falciparum parasites(11-13). In a recent study, researchers at the Harvard School of Public Health sequenced 18 geographically diverse P. falciparum strains (Figure 2)(14). This work resulted in the identification of over 46,000 SNPs in the P. falciparum genome.
Figure 2. 18 geographically diverse strains of P. falciparum were selected for sequencing. Sequence data derived from these strains were used for SNP identification. Courtesy of Science Magazine(14).
According to Dr. Sarah Volkman at the Harvard School of Public Health, over 112,000 SNPs in the P. falciparum genome have been identified by international and domestic groups. A large-scale effort is underway to map the genetic diversity of the P. falciparum parasite and find SNPs that will serve as markers for phenotypes, such as drug resistance. Results from genetic variation studies can be translated into clinical practice through the development of SNP-based molecular diagnostic tests for drug resistance.
DIAGNOSTIC APPLICATIONS
Molecular Diagnosis of Drug Resistance
The overall goal of malaria research discovery efforts is to generate tools that can be used in the field for accurate diagnosis, prevention and treatment of malaria. For example, a diagnostic tool for the detection of drug-resistant malaria strains would help to inform treatment decisions. As a result, much research has been focused on the identification of SNPs that are associated with drug resistance in malaria (see (15) for review). Table 1 briefly summarizes a few of the well-characterized genes and mutations that are associated with drug resistance.
Table 1. P. falciparum genes associated with drug resistance.
Following the discovery of SNPs that are associated with drug resistance, genetic analysis tools must be developed to detect the SNP markers with high specificity and sensitivity. Real-time PCR, restriction fragment length polymorphism and sequencing technologies can be used to identify the SNP markers in endemic countries. Molecular barcoding assays have been developed, which enable investigators to look for specific subsets of SNP markers. Investigators analyzed clinical samples from south eastern Iran for polymorphisms in the pfcrt and pfmdr1 genes, which are associated with chloroquine and multi-drug resistance(26). Similar studies have been conducted in India, Papua New Guinea, Senegal, Ghana and others(27-30). Field-based clinicians can utilize this genetic information to inform drug treatment regimens for individual patients.
Images courtesy of drugs.com (chloroquine and mefloquine).
Genetic analysis tools can also be used to monitor drug resistance at a population level.
SURVEILLANCE AND PREVENTION
Genetic Epidemiology and Population-Based Drug Resistance Monitoring
Widespread resistance to antimalarial medication is a major threat to public health in malaria endemic countries. As shown in Figure 3 below, a high correlation between malaria transmission and drug resistance is shown.
Figure 3. Yellow-highlighted areas indicate regions of malaria transmission. Chloroquine, sulfadoxine-pyrimethamine, and mefloquine resistance areas are marked. Courtesy of the World Health Organization, World Malaria Report, 2005.
A recent study in Malawi highlights the need for a global surveillance network for monitoring drug resistance in P. falciparum(31). Fifteen years ago, Malawi became the first African country to replace chloroquine with a combination therapy of sulfadoxine/ pyrimethamine (SP) for first-line treatment of malaria. This public health policy recommendation was a response to increasing chloroquine resistance in Malawi. Since that time, sulfadoxine/ pyrimethamine drug resistant strains have emerged (Figure 4).
Figure 4. Sulfadoxine/pyrimethamine treatment failure rates in Malawi during the 1998-2002 timeframe. Significant percentages of early and late treatment failures are found throughout the country. Image courtesy of Global Atlas of Infectious Disease.
Results from a 2006 clinical trial demonstrated a significant reversal of the drug resistant strains in Malawi. Investigators found 99% efficacy for chloroquine treatment, and only 22% efficacy for sulfadoxine/ pyrimethamine combination therapy. These findings demonstrate that the genetic variation within the P. falciparum population has evolved under drug pressure. It is likely that similar scenarios exist in other malaria endemic countries. These results highlight the need for improved genetic surveillance mechanisms to inform drug treatment policies.
According to Dr. Mark Perkins, Chief Scientific Officer of the Foundation for Innovative New Diagnostics, genetic analysis tools are highly relevant for the surveillance of drug resistance in P. falciparum. DNA microarrays or RT-PCR assays can be used to genotype P. falciparum strains and detect drug resistance SNP markers. Based on this information, an epidemiological profile of drug resistance can be created for a specific geographic region. All drug resistance profiles could be shared in a global genetic surveillance and mapping database(32). In this situation, genetic analysis tools would be used to inform public health policy recommendations regarding drug regimens in malaria endemic countries.
