Scientists Develop Method to Map Spread of Malarial Drug
Resistance
By Jason Socrates
Bardi
Scientists at The Scripps Research Institute (TSRI), Harvard
University, and the Genomics Institute of the Novartis Research
Foundation have found a way to use a relatively new but readily
available technology to quickly detect markers in the DNA
of the most deadly type of malaria pathogen.
The technology could enable scientists and public health
workers to identify the particular strain of malaria during
an outbreak and determine if it is drug resistant or not.
"One of the reasons for the resurgence of malaria in Africa
and in other parts of the world is the spread of drug resistance,"
says Assistant Professor Elizabeth Winzeler, who is in the
Department of Cell Biology at TSRI and the lead author of
the study described in the latest issue of the journal Science.
The work should make it easier to follow the spread of drug
resistance around the world and assist health ministries in
countries where malaria is a problem to come up with strategies
to thwart this spread. Malaria is a nasty and often fatal
disease, which may lead to kidney failure, seizures, permanent
neurological damage, coma, and death. There are four types
of Plasmodium parasites that cause the disease, of
which falciparum is the most deadly.
Despite a century of effort to globally control malaria,
the disease remains endemic in many parts of the world. With
some 40 percent of the world's population living in these
areas, the need for more effective vaccines is profound. Worse,
strains of Plasmodium falciparum resistant to drugs
used to treat malaria have evolved over the last few decades.
The specter of drug resistance is particularly worrisome
because drug resistance can spread through the mating of Plasmodium
parasites. And drug-resistant Plasmodium falciparum
is more deadly and more expensive to treat. Worse, a drug-resistant
strain could lead to the re-emergence of malaria in parts
of the world where it no longer exists—except for the
occasional imported case—such as the United States.
A New Hope Against a Global Scourge
One of the best tools for fighting any infectious disease
is to track it and fight it where it occurs. And one of the
best ways to determine the origin of a particular malaria
infection and to map the spread of infection is to identify
what are called single nucleotide polymorphisms (SNPs).
Polymorphisms, the genetic variability among various isolates
of one organism, are responsible for drug resistance in malaria
pathogens. In order to follow the spread of drug resistance
around the world, one needs to look at how these markers spread
as well.
In the past, if scientists wanted to detect SNPs, they would
pick one particular gene and sequence it, a time-consuming
process. For instance, finding enough polymorphisms to map
the gene mutation responsible for resistance to the drug chloroquine,
one of the traditional drugs given to patients with malaria,
took several years and millions of dollars to determine.
"Now," says Winzeler, "we have demonstrated that you can
detect thousands of SNPs all at the same time by doing a simple
reaction."
The reaction involves taking DNA from the malaria parasite,
chopping it into fragments, and plopping the mixture of fragmented
DNA on a "gene chip"— a glass or silicon wafer that has
thousands of short pieces of DNA attached to it.
DNA chips have become a standard tool for genomics research
in the last couple of years, and scientists can quite easily
put a large number of different oligonucleotide pieces—even
all the known genes in an organism—on a single chip.
When applying a sample that contains DNA to the chip, genes
that are present in the sample will "hybridize" or bind to
complementary oligonucleotides on the chip. By looking to
see which chip oligonucleotides have DNA bound, scientists
know which genes were being expressed in the sample.
But Winzeler used this technology in a novel way. She compared
the DNA of Plasmodium falciparum parasites that were
resistant to drugs to those that were not and used the differences
in the readouts of the gene chips to determine where the SNPs
were. Nobody had ever used a gene chip in this way before.
Nor did such a chip exist. Winzeler worked with researchers
at the Genomics Institute of the Novartis Research Foundation
to create one just for this purpose.
Using putative malaria genes that were identified in the
international malaria genome effort, Winzeler took sequences
representing 4,000 distinct pieces of these genes on chromosome
2 of the Plasmodium falciparum genome and had a gene
chip constructed.
"Having this type of technology and the genome sequenced
allows us to look at the genome in a whole new way," says
Winzeler. "If you start doing longitudinal studies after you
introduce a new drug, you might be able to identify the drug
targets or the mechanisms of resistance. If you can start
finding the mutations that are associated with drug resistance,
then that tells you how to treat patients in the field."
The new technology should also make it possible to do similar
research with other organisms, characterizing genetic variability
and perhaps conducting population genetics as well. With population
genetics, scientists could quickly determine how similar different
genomes are to each other and generate estimates of a pathogen's
age or its pattern of spread.
Winzeler found that most of the SNPs were in the DNA of
genes that coded for membrane-associated proteins, which is
to be expected, since these are the proteins that are on the
outer surface of the cell and will endure the greatest selective
pressure exerted by host immune systems and drugs.
Significantly, she also found that a number of genes of
unknown function were also high in SNPs, which could mean
that these unknown genes are also under selective pressure.
"These could represent genes that have important functions
in parasite viability or virulence and that warrant further
functional characterization," she concludes.
The article, "Excess Polymorphisms in Genes for Membrane
Proteins in Plasmodium falciparum" was authored by
Sarah K. Volkman, Daniel L. Hartl, Dyann F. Wirth, Kaare M.
Nielsen, Mehee Choi, Serge Batalov, Yingyao Zhou, David Plouffe,
Karine Le Roch, Ruben Abagyan, and Elizabeth A. Winzeler and
appears in the October 4, 2002 issue of the journal Science.
This work was supported by the National Institutes of Health;
the Burroughs Wellcome Fund New Initiatives in Malaria Research
Award; the Ellison Medical Foundation, Program in Career Development,
Research and Training in Global Infectious Diseases; the ExxonMobil
Program on Malaria in Africa; and a new scholars award from
the Ellison Medical Foundation.
The authors of the paper are affiliated with the following
institutions: Harvard University; University of Tromso, Norway;
the Genomics Institute of the Novartis Research Foundation;
and The Scripps Research Institute.
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