A team of U.S. and African medical researchers has developed a molecular marker that can be used to diagnose individuals with and survey populations for malaria parasites that are resistant to the drug chloroquine. The marker may not only help doctors select the best therapy for their patients, it may also assist public health officials determine country-wide treatment guidelines.
The results of their study, reported in this week's New England Journal of Medicine, puts a confirmatory clinical stamp on the recent laboratory discovery that tiny mutations in a single gene of the malaria parasite confer resistance to the drug. In the new study, the marker was found 100 percent of the time in clinical cases of chloroquine-resistant malaria.
A safe, inexpensive and highly effective treatment, chloroquine was the mainstay antimalarial drug worldwide in the latter half of the 20th century until overuse pressured Plasmodium falciparum, the most deadly malaria parasite, to develop ways to evade its effects. Doctors in South America and Southeast Asia have largely given up using the drug. Resistant parasites continue to spread, especially in Africa where 90 percent of malaria deaths, primarily among young children, now occur. Although the problem also seriously undercuts malaria control efforts in Africa-most dramatically in sub-Saharan Africa-chloroquine remains the treatment of choice for many African countries because affordable alternatives do not exist, and partial immunity among older children and adults is widespread and helps the drug work.
The NEJM study, led by Christopher V. Plowe, M.D., Abdoulaye Djimde, Pharm.D., and their colleagues at the University of Maryland School of Medicine in Baltimore, the University of Mali in Bamako, and the National Institute of Allergy and Infectious Diseases (NIAID), is the first of several ongoing field studies to confirm laboratory findings published last October by Thomas E. Wellems, M.D., Ph.D., and his colleagues in NIAID's Laboratory of Parasitic Diseases. The NIAID group reported that small changes in the pfcrt gene of chromosome 7 of P. falciparum associate completely with chloroquine resistance in parasite lines from Asia, Africa and South America.
"Our clinical data strongly support that this pfcrt mutant is responsible for chloroquine resistance," states Dr. Djimde, "but the level of resistance may be modulated by other factors or other genes."
"The clinical validation of this laboratory marker is good news for diagnosing chloroquine resistance in the field as well as in clinics and hospitals," says NIAID Director Anthony S. Fauci, M.D. "Importantly, public health officials in malaria-endemic countries may use this tool to survey their populations for increases or decreases in chloroquine-resistant parasites, helping them make informed decisions about front-line malaria therapy."
In addition, Dr. Djimde notes, the tool can also be used at the individual level to diagnose chloroquine-resistant malaria in non-immune individuals, for example, travelers or people who live in places where malaria occurs only sporadically, such as deserts or highlands within endemic countries. Non-immune individuals are at greater risk for the severe complications of malaria when they are infected.
Currently, it takes 14 days to diagnose a chloroquine-resistant infection in an individual, and two to three days to detect resistance in laboratory strains of the parasite. The molecular tool developed by the University of Maryland and University of Mali groups, which relies in part on the ultrasensitive diagnostic technique called polymerase chain reaction (PCR), takes only a few hours. "Our ultimate goal," says Dr. Djimde, " is to develop a biochemical method to detect the presence of the mutant gene in a few minutes, but we're not there yet."
The investigators carried out their study in the towns of Mopti and Bandiagara in central eastern Mali, areas with a low level of resistance to chloroquine. They invited community members age 2 years or older who had malaria symptoms to be examined by a physician at their NIAID-funded clinic. Individuals who met the study criteria and agreed to participate in the study received chloroquine treatment and were followed by the study team for 14 days afterwards.
Dr. Plowe and his colleagues collected blood samples before and after treatment and analyzed them for specific mutations in two different P. falciparum genes, pfcrt and pfmdr 1, based on earlier work suggesting that these genes might harbor mutations important to chloroquine resistance.
Among patients in the study, they found the parasite T76 pfcrt mutation in 100 percent of 60 people with infections that failed to respond to chloroquine treatment versus 41 percent of 116 infected people who were randomly sampled prior to receiving treatment. The Y86 pfmdr 1 mutation had a statistically significant but weaker link with chloroquine resistance. Based on their findings, the researchers believe that the pfcrt mutant is responsible for chloroquine resistance but that the pfmdr 1 mutant may help modulate the level of resistance.
Some people who carried the resistant parasite did clear their infections after chloroquine therapy. To determine what role prior immunity may have played in this outcome, the investigators compared infection clearances in children younger than 10 years old to those among the older individuals. In the younger group, 69 percent of pre-treatment infections with the T76 pfcrt mutation failed chloroquine treatment compared with only 34 percent of such pre-treatment infections in the older group.
One current research focus, says Dr. Djimde, is to understand which immunological events contribute to clearing resistant parasites. Using their new tool, they will compare patients who can clear the resistant parasites with those who cannot, and examine the immunological differences in these two populations.
"Malaria is the number one killer in Mali and one of the leading causes of death in Africa," says Dr. Djimde. "Our goal in Mali is to do applied research to help our health authorities combat malaria and to ease the burden of the disease on our people."
NIAID is a component of NIH. NIAID supports basic and applied research to prevent, diagnose, and treat infectious and immune-mediated illnesses, including HIV/AIDS and other sexually transmitted diseases, tuberculosis, malaria, autoimmune disorders, asthma and allergies.
1. A Djimde et al. A molecular marker for chloroquine-resistant falciparum malaria. New England Journal of Medicine 344:257-63 (2001).
2. D Warhurst. A molecular marker for chloroquine-resistant falciparum malaria. New England Journal of Medicine 344:299-301 (2001).
3. DA Fidock et al. Mutations in the Plasmodium falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Molecular Cell 6(4): 861-71 (2000).
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