Independent Origin of Plasmodium falciparum Antifolate Super-Resistance, Uganda, Tanzania, and Ethiopia

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Michael Alifrangis1, Sidsel Nag1

, Mette L. Schousboe, Deus Ishengoma, John Lusingu, Hirva Pota, Reginald A. Kavishe, Richard Pearce, Rosalynn Ord, Caroline Lynch, Seyoum Dejene, Jonathan Cox, John Rwakimari, Daniel T.R. Minja, Martha M. Lemnge, and Cally Roper

Author affiliations: Centre for Medical Parasitology at Department of International Health, Immunology, and Microbiology, Faculty of Health and Medical Science, University of Copenhagen and at Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet),; Copenhagen, Denmark (M. Alifrangis, S. Nag, M.L. Schousboe); Tanga Research Centre, Tanzania (D. Ishengoma, J. Lusingu, D.T.R. Minja, M.M. Lemnge);London School of Hygiene and Tropical Medicine, London, UK (H. Pota, R. Pearce, R. Ord, C. Lynch, J. Cox, C. Roper); Kilimanjaro Christian Medical University College, Moshi, Tanzania (R.A. Kavishe); Kilimanjaro Clinical Research Institute, Moshi (R.A. Kavishe); Médécins Sans Frontières, Humera, Ethiopia (S. Dejene); Ministry of Health of Uganda, Kampala, Uganda (J. Rwakimari)

Abstract

Super-resistant Plasmodium falciparum threatens the effectiveness of sulfadoxine–pyrimethamine in intermittent preventive treatment for malaria during pregnancy. It is characterized by the A581G Pfdhpsmutation on a background of the double-mutant Pfdhps and the triple-mutant Pfdhfr. Using samples collected during 2004–2008, we investigated the evolutionary origin of the A581G mutation by characterizing microsatellite diversity flanking Pfdhps triple-mutant (437G+540E+581G) alleles from 3 locations in eastern Africa and comparing it with double-mutant (437G+540E) alleles from the same area. In Ethiopia, both alleles derived from 1 lineage that was distinct from those in Uganda and Tanzania. Uganda and Tanzania triple mutants derived from the previously characterized southeastern Africa double-mutant lineage. The A581G mutation has occurred multiple times on local Pfdhps double-mutant backgrounds; however, a novel microsatellite allele incorporated into the Tanzania lineage since 2004 illustrates the local expansion of emergent triple-mutant lineages.

Controlling and reducing malaria requires a combination of vector control measures and administration of antimalarial drugs as prophylaxis or treatment (1). The widespread use of antimalarial drugs has resulted in the emergence of resistant Plasmodium falciparum, recurrently exposing persons in malaria-endemic regions to an unacceptably high risk for treatment failures (2).

Highly chloroquine-resistant parasites spread from Asia in the 1960s and led to devastating rates of malaria-related death in Africa starting in the late 1980s, gradually forcing affected countries to replace chloroquine with sulfadoxine–pyrimethamine (SP) (3–5). The effectiveness of SP did not last long. In fact, retrospective analysis indicated that pyrimethamine-resistant parasites were present in sub-Saharan Africa before SP was implemented as first-line treatment, probably because pyrimethamine as monotherapy had been used in Asia during the 1960’s and 1970’s (6–8). Resistance to sulfadoxine also soon emerged (9), and the combination of pyrimethamine- and sulfadoxine-resistant parasites led to severe and widespread SP treatment failure (10–12). As a consequence, affected countries were once again forced to change their drug policies (13) and have now adopted artemisinin-based combination therapies as first-line treatment for uncomplicated malaria. Yet, SP is still recommended for use as intermittent preventive treatment in pregnant women (SP-IPTp) and infants (SP-IPTi) (14,15). Also, seasonal malaria chemoprevention applies SP in combination with amodiaquine (16). Use of SP for prevention in many countries of sub-Saharan Africa, where clinical failure after SP treatment has been reported, underscores the need for effective surveillance of its protective efficacy and for monitoring of the development and spread of SP resistance in P. falciparum populations.

The molecular basis of SP resistance is a combination of single-nucleotide polymorphisms (SNPs) in 2 distinct genes coding for the target enzymes of SP. The enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are targeted by pyrimethamine and sulfadoxine, respectively (17). High-level pyrimethamine resistance is generally encoded by 3 mutations in the Pfdhfr gene, coding for substitutions: N51I, C59R, and S108N (18); the molecular basis of sulfadoxine resistance is caused by substitutions S/A436F, A437G, K540E, A581G, and A613S/T in a variety of combinations in DHPS (19).

The most prevalent genotype in eastern Africa is a combination of the Pfdhfr triple mutant (51I, 59R, and 108N, denoted as IRN) combined with thePfdhps double mutant (S436, 437G, 540E, A581, and A613, denoted as SGEAA). Together, this combination of SNPs is referred to as the “quintuple” mutant Pfdhfr/Pfdhps genotype and is associated with high risk for SP treatment failure (17) and results in limited protective value of SP-IPTi (20). Accordingly, the World Health Organization (WHO) recommends that SP-IPTi should be implemented only when the prevalence of the K540E mutation (and thus the quintuple mutant) is <50% (14).

More recently, an alanine to glycine mutation at codon position 581 in Pfdhps has emerged that, in combination with the Pfdhfr triple-mutant allele IRN, was shown to confer higher level resistance (21). This combination, referred to as the “sextuple Pfdhfr/Pfdhps mutant genotype” or the “super-resistant genotype” (22), is associated with reduced SP-IPTp efficacy by 1) a reduction in the protection period of SP-IPTp from 4 weeks to 2 weeks (23); 2) increased parasitemia attributed to competitive facilitation (23); 3) increased risk for severe malaria in the offspring (24); and 4) low birthweight in newborns from mothers undergoing SP-IPTp in Tanzania (25). Consequently, WHO recommendations concerning the use of SP-IPTp base the threshold on 2 mutations: SP-IPTp should be discontinued if the prevalence of the K540E mutation is >95% and the A581G mutation is >10% (20). No threshold in the prevalence of molecular markers of resistance has been set with regard to seasonal malaria chemoprevention (15,16).

Maps collating all published data from molecular surveillance of Pfdhfr and Pfdhps mutations (22) indicate 3 main foci of super-resistant parasites: 1 in northern Tanzania (26); a second in southwestern Uganda, Rwanda, and bordering areas of Democratic Republic of Congo (27–29); and a third in western Kenya (30). Prevalence of A581G also is high in Ethiopia and northern Sudan, where it again occurs as the Pfdhps triple-mutant allele SGEGA but in combination with a Pfdhfr double-mutant allele 51I-108N.

Assessments of microsatellite variation linked to Pfdhps have shown that limited microsatellite diversity flanking the SGEAA double mutants compared with the SAKAA wild types. Two SGEAA lineages were discovered in eastern Africa: 1 prevailing in northeastern Africa (Ethiopia and Sudan) and the other throughout southeastern Africa. Both lineages derived from independent ancestry (10). Here we apply the same approach, using the same microsatellite loci, to determine the ancestry and possible relationship between the double SGEAA and triple SGEGA alleles in Ethiopia, Uganda, and Tanzania. By focusing on microsatellite variation linked to Pfdhps, we can explore whether the emergence of the SP-IPT-threatening SGEGA triple mutants in Ethiopia, Uganda, and Tanzania derive from local SGEAA alleles or are being imported.